KR20110129945A - Nucleic acid sequences encoding and compositions comprising ige signal peptide and/or il-15 and methods for using the same - Google Patents

Abstract

Fusion proteins and nucleic acid molecules encoding fusion proteins are described. Fusion proteins comprising non-IL-15 signal peptides linked to IL-15 protein sequences and fusion proteins comprising IgE signal peptides linked to non-IgE protein sequences are described. Vectors comprising such nucleic acid molecules; And attenuated live pathogens encoding recombinant vaccines and fusion proteins, as well as host cells comprising such vectors, are described and methods using them are described. The immunomodulatory effects after delivery of IL-15 and CD40L with or without immunogen are described, and methods using such compositions and compositions thereof and various nucleic acid molecules used to deliver such proteins are described.

Description

Nucleic acid sequences encoding and/or IL-15 encoding nucleic acid sequences and compositions comprising the same, and methods for using the same {Nucleic acid sequences encoding and compositions comprising IgE signal peptide and/or IL-15 and methods for using the same}

The present invention relates to improved vaccines, improved methods and improved immunotherapeutic compositions and improved immunotherapeutic methods for prophylactically and therapeutically immunizing individuals against immunogens.

Immunotherapy is to give a desired therapeutic effect by modulating a person's immune response. When administered to a subject, an immunotherapeutic agent refers to a composition that, when administered to a subject, sufficiently modulates the subject's immune system to ultimately reduce symptoms associated with an undesired immune response or increase a desired immune response and ultimately relieve the symptoms. In some cases, immunotherapy is part of a vaccination protocol in which a subject is administered a vaccine that exposes the subject to an immunogen and causes the subject to generate an immune response. In this case, the immunotherapeutic agent increases the immune response and/or selectively enhances a portion of the immune response (e.g., cellular immunity or humoral immunity) desired to treat or prevent a particular symptom, infection or disease. .

Vaccines are useful for immunizing individuals against antigens associated with allergens, pathogen antigens or cells involved in human disease. Antigens associated with cells involved in human diseases include cancer-related tumor antigens and antigens associated with cells involved in autoimmune diseases.

In designing this vaccine, it has been recognized that a vaccine that produces a target antigen in cells of vaccinated individuals is effective in inducing cellular immunity of the immune system. In particular, live attenuated vaccines, recombinant vaccines using non-toxic vectors, and DNA vaccines each induce the production of antigens in cells of vaccinated individuals, thereby inducing cellular immunity of the immune system. On the other hand, killed or inactivated vaccines and subunit vaccines containing only proteins, they induce humoral responses, but are not good for induction of cellular immune responses.

Cellular immune responses often provide protection against pathogen infections and are required to provide effective immune mediated treatment for the treatment of pathogen infections, cancer or autoimmune diseases. Thus, vaccines that produce target antigens in the cells of vaccinated individuals, such as live attenuated vaccines, recombinant vaccines using non-toxic vectors, and DNA vaccines, are often desirable.

Such vaccines are often effective in immunizing individuals prophylactically or therapeutically against pathogen infections or human diseases, but there is a need for improved vaccines. There is a need for compositions and methods that produce an enhanced immune response.

In addition, while some immunotherapeutic agents are useful for modulating the immune response in patients, there is still a need to improve immunotherapeutic compositions and methods.

Gene therapy is the delivery of a gene to a patient who needs or can benefit from the protein encoded by the protein. New strategies have been developed to deliver proteins that do not have the gene in question to produce a protein that is sufficient and/or fully functional in an individual. Thus, gene therapy completely compensates for the deficiency of a fully functioning endogenous protein. In some gene therapy strategies, a construct designed to produce a therapeutically effective amount of a protein is used to provide a therapeutically effective protein to a patient. Gene therapy provides another method for delivering protein therapeutics. Still, there is a need to improve gene therapy compositions and methods.

In addition to the direct administration of the nucleic acid molecule to an individual, the protein is often delivered. Production of these proteins by recombinant methods is often the most efficient way to make them. Still, there is a need for improved proteins to construct compositions and methods.

The present invention includes a nucleic acid sequence encoding a fusion protein comprising a nucleic acid sequence encoding an immunogen and a non-IL-15 signal sequence linked to an IL-15 protein sequence, and optionally a nucleic acid molecule comprising a nucleic acid sequence encoding CD40L Recombinant vaccines; And administering the recombinant vaccine to the subject.

The present invention provides an attenuated biopathogen comprising a nucleic acid sequence encoding a fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein sequence and optionally a nucleic acid molecule comprising a nucleic acid sequence encoding CD40L; And a method of immunizing a subject and a method of immunizing a subject against a pathogen comprising administering to the subject the attenuated biopathogen.

The present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an IL-15 protein and a nucleic acid sequence encoding a CD40L protein and optionally a nucleic acid sequence encoding an immunogen.

The present invention is a composition comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an IL-15 protein and a nucleic acid molecule comprising a nucleic acid sequence encoding a CD40L protein, and optionally a nucleic acid sequence encoding an immunogen in either or both of the nucleic acid molecules. It is about.

The present invention relates to a method of modulating an immune response in an individual comprising administering to the individual a composition comprising one or more nucleic acid molecules comprising a nucleic acid sequence encoding an IL-15 protein and a nucleic acid sequence encoding CD40L. will be. Various nucleic acid sequences encoding various different proteins may be present on the same nucleic acid molecule and/or on different nucleic acid molecules or on both nucleic acid molecules.

The present invention relates to an immunogen in an individual comprising administering to the individual a composition comprising a nucleic acid sequence encoding an IL-15 protein and a nucleic acid sequence encoding an immunogen and a nucleic acid sequence encoding CD40L. It relates to a method of inducing an immune response against.

Various nucleic acid sequences encoding various different proteins may exist on the same nucleic acid molecule and/or on different nucleic acid molecules, or in combinations thereof.

The present invention comprises a recombinant vaccine comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen, a nucleic acid sequence encoding an IL-15 protein, and a nucleic acid sequence encoding CD40L, and administering the recombinant vaccine to an individual, It relates to a method of immunizing an individual against an immunogen.

The present invention relates to an attenuated biopathogen comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an IL-15 protein and a nucleic acid sequence encoding CD40L, and a pathogen comprising the step of administering the attenuated biopathogen to an individual. It relates to a method of immunizing an individual against.

The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence, wherein the IgE signal peptide and the non-IgE protein sequence originate from the same animal species. will be.

The present invention includes an in vitro host cell culture comprising an expression vector operable in a host cell comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence, the nucleic acid molecule, and the vector. It relates to a host cell.

The present invention provides a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to a non-IgE protein sequence operably linked to a regulatory factor required for expression and an immunogen operably linked to a regulatory factor required for expression. It relates to a nucleic acid molecule comprising an encoding nucleic acid sequence.

The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to a non-IgE protein sequence and a composition comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen, wherein A nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein is not identical to a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen.

The present invention relates to an isolated fusion protein comprising an IgE signal peptide linked to a non-IgE protein sequence.

The present invention relates to a method of modulating an immune response in an individual comprising administering to the individual a composition comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to an immunomodulatory protein. will be.

The present invention comprises the step of administering to the individual a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to an immunomodulatory protein and a nucleic acid sequence encoding an immunogen, an immune response against an immunogen in an individual It relates to how to induce. Various coding sequences for different proteins can be on the same nucleic acid molecule and/or on different nucleic acid molecules.

The present invention comprises a recombinant vaccine comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen and a nucleic acid sequence encoding a fusion protein comprising an IgE signal sequence linked to an immunomodulatory protein, and administering the recombinant vaccine to an individual. It relates to a method of immunizing an individual against an immunogen.

The present invention comprises the step of administering to an individual an attenuated biopathogen comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal sequence linked to an immunomodulatory protein and the attenuated biopathogen. , To a method of immunizing an individual against pathogens.

The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to an IL-15 protein sequence, a vector comprising the nucleic acid molecule, and a host cell comprising the vector.

The present invention relates to a fusion protein comprising an IgE signal peptide linked to an IL-15 protein sequence.

The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to an IL-15 protein sequence and a composition comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen. Optionally, the nucleic acid sequence encoding CD40L may be present in a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein and/or immunogen or in a separate nucleic acid molecule.

The present invention comprises administering to a subject a composition comprising one or more nucleic acid molecules comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to an IL-15 protein and optionally a nucleic acid sequence encoding CD40L. , To a method of modulating the immune response in an individual. Various nucleic acid sequences encoding various different proteins may be present in the same nucleic acid molecule and/or different nucleic acid molecules or both.

The present invention provides a composition comprising at least one nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal peptide linked to an IL-15 protein sequence, a nucleic acid sequence encoding an immunogen, and a nucleic acid sequence encoding CD40L. It relates to a method of inducing an immune response against an immunogen in a subject, comprising the step of administering to. Various nucleic acid sequences encoding a variety of different proteins may exist on the same nucleic acid molecule and/or on different nucleic acid molecules or in combinations thereof.

Justice

The term “target protein” as used herein refers to the peptides and proteins encoded by the genetic constructs of the invention that act as target proteins for an immune response. The terms “target protein” and “immunogen” are used interchangeably and refer to proteins capable of eliciting an immune response. A target protein is an immunogenic protein that shares one or more epitopes with a protein originating from an undesired cell type, such as a pathogen or a cancer cell or a cell involved in an autoimmune disease in which an immune response is required. The immune response to the target protein protects and/or treats the individual from a specific infection or disease associated with the target protein.

As used herein, the term “gene construct” refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a target protein or immunoregulatory protein. The coding sequence includes initiation and termination signals operatively linked to regulatory factors including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered.

As used herein, the term “expressible form” refers to a gene that, when present in a cell of an individual, causes the coding sequence to be expressed, including the necessary regulatory factors operably linked to a coding sequence encoding a target protein or immunomodulatory protein. Refers to the creation.

As used herein, the term “sharing of an epitope” refers to a protein comprising one or more epitopes that are substantially similar or identical to those of other proteins.

The term “substantially similar epitope” as used herein refers to an epitope that is not identical to an epitope of a protein, but has a structure that elicits a cellular or humoral immune response that cross-reacts with the protein.

The term “intracellular pathogen” as used herein refers to a virus or pathogenic organism in which at least a portion of its reproductive or life cycle is present in a host cell and produces or causes the pathogen protein to be produced therein.

The term “hyperproliferative disease” as used herein refers to diseases and disorders characterized by hyperproliferation of cells.

The term “hyperproliferative associated protein” as used herein refers to a protein associated with hyperproliferative disease.

The term “immunomodulatory protein” as used herein refers to a protein that modulates the immune system of a person to which the immunoregulatory protein is delivered. Examples of immunomodulatory proteins include: IL-15, CD40L, TRAIL; TRAILrecDRC5, TRAIL-R2, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, F461811or MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, CD30, CD153 c-jun, Sp-1, Apl, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, NIK, SAP K, SAP1, JNK2, JNK1B2, JNK1B1, JNK2B2, JNK2B1, JNK1A2, JNK2A1, JNK3A1, JNK3A1 , NF-kappa-B2, p49 splicing form, NF-kappa-B2, plOO splicing form, NF-kappa-B2, p105 splicing form, NF-kappa-B 50K chain precursor, NFkB p50, human IL-1a, human IL-2, human IL-4, mouse IL-4, human IL-5, human IL-10, human IL-15, human IL-18, human TNF-a, human TNF-P, Human Interleukin 12, MadCAM-1, NGF IL-7, VEGF, TNF-R, Fas, CD40L, IL-4, CSF, G-CSF, GM-CSF, M-CSF, LFA-3, ICAM-3, ICAM -2, ICAM-1, PECAM, P150.95, Mac-1, LFA-1, CD34, RANTES, IL-8, MIP-la, E-selectone, CD2, MCP-1, L-selectone, P -Selectone, FLT, Apo-1, Fas, TNFR-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4 (TRAIL), DR5, KILLER, TRAIL-R2, TRICK2 , DR6, ICE, VLA-1 and CD86 (B7.2).

summary

The invention derives from the following findings: 1) A fusion comprising an IL-15 protein sequence linked to an IL-15 protein as a “cleaved” IL-15 protein or with a non-IL-15 signal peptide, in particular an IgE signal peptide. In the absence of the IL-15 signal peptide, whether expressed as a protein, the level of IL-15 protein expression is higher. IL-15 proteins deficient in the IL-15 signal peptide, whether "cleaved" IL-15 protein or a non-IL-15 signal peptide, especially a fusion protein comprising an IL-15 protein sequence linked to an IgE signal peptide, are specifically, As an immunomodulatory protein, it is useful in vaccines and constructs for the delivery of the IL-15 protein. 2) Vaccines and immunomodulatory compositions involved in the delivery of IL-15 in combination with CD40L are particularly useful. 3) Fusion proteins comprising IgE signal peptides promote enhanced expression and are particularly useful in vaccines and gene therapy for the delivery of proteins such as protein production, immunomodulatory proteins. In some preferred embodiments, the present invention comprises a protein or a human IL-15 coding sequence deficient in the IL-15 signal peptide, preferably a human IL-15 coding sequence deficient in the IL-15 Kozak region and the untranslated region. Vectors, vaccines and immunomodulatory compositions and methods comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein provided with a non-IL-15 signal peptide, in particular an IgE signal peptide are provided. The IL-15 coding sequence preferably lacks the IL-15 signal sequence and preferably the IL-15 Kozak region and the untranslated region. In some preferred embodiments, the present invention 1) encodes a fusion protein comprising an IL-15 protein sequence to which an IL-15 signal peptide is bound or an IL-15 protein sequence linked to a non-IL-15 signal peptide such as an IgE signal peptide. A vector, vaccine and immunomodulatory composition and method comprising a nucleic acid molecule comprising a nucleic acid sequence and a nucleotide sequence encoding human CD40L in combination therewith are provided. The IL-15 coding sequence preferably lacks the IL-15 signal sequence and preferably the IL-15 Kozak region and the untranslated region. In some preferred embodiments, the present invention provides a vector, vaccine and a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein in which the IgE signal peptide is linked to a non-IgE protein sequence, preferably a human IL-15 protein sequence. Immunomodulatory compositions and methods are provided.

Fusion protein comprising an IgE signal sequence linked to a non-IgE protein and a gene construct encoding the same

Accordingly, one general aspect of the present invention relates to a fusion protein comprising an IgE signal sequence linked to a non-IgE protein and to the use of the construct in gene constructs and expression vectors encoding the same, vaccines and immunomodulatory compositions.

According to some embodiments, a composition is provided comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an IgE signal sequence operably linked with a non-IgE protein sequence.

The properties of the non-IgE protein depend on the intended use of the construct. For example, for gene therapy embodiments, a protein sequence is a sequence of a protein of interest, such as a protein for which the patient does not have a protein that functions in sufficient amounts or in full. Examples of this type of protein of interest include enzymes such as DNAse, growth factors such as growth hormone (human, bovine, pig), coagulation factors, insulin, dystrophin, and the like. The protein of interest can also be a protein that provides a therapeutic benefit when expressed in a patient, examples of which are erythropoietin, IL-2, GM-CSF and TPA. In some embodiments, the non-IgE protein sequence is an immunogen. This construct is useful in vaccines in which expression of an immunogen is provided as a target for an immune response. In some embodiments, the non-IgE protein sequence is an immunomodulatory protein. This construct is not only useful in a vaccine in which the expression of an immunogen is provided as a target for an immune response, but also provides a desired effect to the immune system of a patient or provides a specific aspect in which the immune system is upregulated or downregulated depending on the symptoms of the patient to be treated It is useful in the provided immunomodulatory composition. Immunomodulators that up-regulate the immune system are useful, for example, in treating patients suffering from immunosuppression or infectious diseases, whereas immunomodulatory agents that down-regulate the immune system are, for example, autoimmune diseases where immune suppression is desired, organ transplants, tissues. It is useful for treating patients undergoing transplantation or cell therapy. In some embodiments, the IgE signal sequence is linked to a non-IgE protein sequence for use in a system where production of IgE protein is desired. In a preferred embodiment, the IgE signal peptide is from an animal of the same species as the protein sequence to which it is linked. In a preferred method, the animal to which the construct is administered is the same species as the animal from which the IgE signal peptide and protein sequences are derived. This fusion protein is believed to be non-immunogenic.

In some embodiments, a composition comprising a construct comprising a coding sequence of an IgE signal linked to a non-IgE protein sequence that is an immunomodulatory protein may also comprise a nucleic acid sequence encoding an immunogen on the same nucleic acid molecule or on a different nucleic acid molecule. have. In general, the immunogens discussed below can be any immunogenic protein, including allergens, pathogen antigens, cancer-related antigens, or antigens linked to cells associated with an autoimmune disease. In a preferred embodiment, the immunogen is a pathogen antigen and most preferably a pathogen selected from the group consisting of HIV, HSV, HCV and WNV.

As noted above, the non-IgE protein sequence is preferably an IL-15 protein, more preferably an IL-15 protein deficient in the IL-15 signal sequence, more preferably an IL-15 signal sequence deficient, IL It is an IL-15 protein that lacks the -15 Kozak region and lacks the IL-15 untranslated sequence. In some preferred embodiments, the composition further comprises a nucleotide sequence encoding CD40L. The nucleotide sequence can be included on the same nucleic acid molecule or on a different molecule as a fusion protein. CD40L can be included in a vaccine composition comprising a coding sequence for an immunogen to provide an improved vaccine. In other embodiments, CD40L can be included in an immunomodulatory composition that does not include a coding sequence for an immunogen, providing an improved immunomodulatory composition.

In some preferred embodiments, the nucleic acid construct is a plasmid. In some preferred embodiments, the nucleic acid molecule is incorporated into a viral vector such as vaccinia, adenovirus or adenovirus related virus or other acceptable viral vector or gene therapy vector useful as a vaccine.

Genetic constructs comprising an IgE signal sequence linked to a non-IgE protein sequence, which is an immunoregulatory protein, can be incorporated directly into an attenuated biopathogen according to some aspects of the invention. Examples of such pathogens useful as vaccines are shown below. In a preferred embodiment, the immunomodulatory protein is IL-15, more preferably an IL-15 protein deficient in the IL-15 signal sequence, more preferably an IL-15 signal sequence, an IL-15 Kozak region, and an IL-15 untranslated. It is a sequence-deficient IL-15 protein. In some embodiments, the attenuated pathogen is further provided with a nucleotide sequence encoding CD40L.

Another aspect of the present invention is a fusion protein comprising an IgE signal sequence operably linked to a non-IgE protein sequence. In some embodiments, the non-IgE protein sequence of the fusion protein is an enzyme. In some embodiments, the non-IgE protein sequence portion of the fusion protein is an immunogen. In some embodiments, the non-IgE protein sequence portion of the fusion protein is an immunomodulatory protein. A preferred non-IgE sequence is an IL-15 protein, most preferably an IL-15 protein lacking the IL-15 signal sequence.

Fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein and a gene construct encoding the same

One general aspect of the present invention relates to a fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein and to the use of the construct in a genetic construct encoding the same and in vaccines and immunomodulatory compositions. Several different aspects and forms are provided in connection with this aspect. In general, IL-15 refers to human IL-15. However, the construct may also refer to IL-15 from other species, such as, for example, dogs, cats, horses, cattle, pigs or sheep.

This aspect of the invention arises from the observation that the sequence in the protein expressed by the native IL-15 mRNA contains a signal or factor that inhibits expression. By removing this inhibitory factor, improved expression is achieved. In a preferred embodiment, the IL-15 coding sequence lacks the coding sequence for the IL-15 signal peptide and preferably another signal protein, such as an IgE signal protein, is provided instead. Moreover, the IL-15 Kozak and untranslated regions are also eliminated to eliminate inhibitory factors. The only IL-15 sequence that the construct preferably contains is an IL-15 sequence that encodes the amino acid sequence of a mature IL-15 protein deficient in the IL-15 signal peptide.

According to some embodiments, a composition is provided comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein. In some preferred embodiments, the fusion protein consists of a non-IL-15 signal sequence linked to an IL-15 protein. In some preferred embodiments, the IL-15 protein lacks the IL-15 signal sequence. In some preferred embodiments, the fusion protein is non-immunogenic compared to the species from which the IL-15 sequence is derived. Thus, non-immunogenic fusion proteins comprising human IL-15 are non-immunogenic in humans.

According to some embodiments, it may comprise a coding sequence for a fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein and also a nucleic acid sequence encoding an immunogen on the same nucleic acid molecule or on a different nucleic acid molecule. Compositions are provided containing constructs that are present. The immunogens generally discussed below can be any immunogenic protein, including allergens, pathogen antigens, cancer-related antigens, or antigens linked to cells associated with an autoimmune disease. In a preferred embodiment, the immunogen is a pathogen antigen, most preferably a pathogen selected from the group consisting of HIV, HSV, HCV and WNV.

In a preferred embodiment, the composition further comprises a nucleotide sequence encoding CD40L. The nucleotide sequence can be included on the same nucleic acid molecule or on a different molecule as a fusion protein. CD40L can be included in a vaccine composition comprising a coding sequence for an immunogen to provide an improved vaccine. In other embodiments, CD40L can be included in an immunomodulatory composition that does not contain a coding sequence for an immunogen to provide an improved immunomodulatory composition.

In some preferred embodiments, the nucleic acid construct is a plasmid. In some preferred embodiments, the nucleic acid molecule is incorporated into a viral vector such as vaccinia, adenovirus, adenovirus related virus or retrovirus, or any other acceptable viral vector or gene therapy vector useful as a vaccine.

Genetic constructs comprising a nucleotide sequence encoding a fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein can be incorporated directly into the attenuated biopathogen according to some aspects of the invention. Examples of such pathogens useful as vaccines are shown below. In a preferred embodiment, human IL-15, preferably lacking the IL-15 signal sequence, is linked to the human IgE signal sequence. In some embodiments, the attenuated pathogen is further provided with a nucleotide sequence encoding CD40L.

One aspect of the invention is a fusion protein comprising a non-IL-15 signal sequence linked to an IL-15 protein sequence. In some preferred embodiments, the fusion protein consists of a non-IL-15 signal sequence linked to an IL-15 protein. In some preferred embodiments, the IL-15 protein lacks the IL-15 signal sequence. In some preferred embodiments, the signal sequence is an IgE signal sequence. The sequence is preferably of human origin. In some preferred embodiments, the fusion protein is non-immunogenic. Non-immunogenic refers to a protein that is non-immunogenic compared to the species from which the IL-15 sequence is derived.

Composition comprising the genetic construct encoding IL-15 and CD40L and methods of using the same

Another general aspect of the invention relates to compositions comprising the genetic constructs encoding IL-15 and CD40L and the use of such constructs in vaccines and immunomodulatory compositions. Several different aspects and forms are provided in connection with this aspect. In general, IL-15 refers to human IL-15. However, the construct may also refer to IL-15 derived from other species such as dogs, cats, horses, cattle, pigs or sheep. IL-15 can be in its native form, i.e. having an IL-15 signal sequence. Preferably, the IL-15 is part of a fusion protein comprising a non-IL-15 signal sequence and most preferably a further lack of the IL-15 signal sequence. In a preferred embodiment, IL-15 is linked to the IgE signal sequence.

According to some embodiments, an isolated nucleic acid molecule comprising a nucleic acid sequence encoding IL-15 and CD40L or a first molecule comprising a nucleic acid sequence encoding IL-15 and a second molecule comprising a nucleic acid sequence encoding CD40L Compositions are provided comprising two different isolated nucleic acid molecules comprising. In some preferred embodiments, the protein comprising IL-15 is non-immunogenic compared to the species from which the IL-15 sequence is derived.

In accordance with some embodiments, compositions are provided comprising a construct comprising a coding sequence for IL-15 and CD40L and a nucleic acid sequence encoding an immunogen on the same nucleic acid molecule or on a different nucleic acid molecule. In general, the immunogens discussed below can be any immunogenic protein, including allergens, pathogen antigens, cancer-related antigens, or antigens linked to cells associated with an autoimmune disease. In a preferred embodiment, the immunogen is a pathogen antigen, most preferably a pathogen selected from the group consisting of HIV, HSV, HCV and WNV.

Compositions comprising the coding sequence for an immunogen are useful as vaccines. Compositions that do not contain a coding sequence for an immunogen may be useful as an immunomodulatory composition. In some embodiments, the protein immunogen also serves as a target for an immune response enhanced by the combination of IL-15 and CD40L.

In some preferred embodiments, the nucleic acid construct is a plasmid. In some preferred embodiments, the nucleic acid molecule is incorporated into a viral vector such as vaccinia, adenovirus, adenovirus related virus or retrovirus, or any other acceptable viral vector or gene therapy vector useful as a vaccine.

Genetic constructs comprising nucleotide sequences encoding IL-15 and CD40L can be incorporated directly into a viable attenuated pathogen according to some aspects of the invention. Examples of such pathogens useful as vaccines are shown below. In a preferred embodiment, human IL-15, preferably lacking the IL-15 signal sequence, is linked to a human IgE signal sequence.

Vaccine and immunomodulatory composition

According to some aspects of the invention, the compositions of the invention comprise a genetic construct comprising the coding sequence for an immunogen and/or immunogenic protein. The composition is delivered to an individual to enhance the immune response to the immunogen by modulating the activity of the individual's immune system. When a nucleic acid molecule encoding an immunoregulatory protein is taken up by a cell of an individual, the nucleotide sequence encoding the immunoregulatory protein is expressed in the cell, whereby the protein is delivered to the individual. An aspect of the invention is a method of delivering the coding sequence of a protein on a single nucleic acid molecule in a composition comprising different nucleic acid molecules encoding one or more various transcription factors or intermediate factors as part of a recombinant vaccine and as part of an attenuated vaccine. Provides.

According to some aspects of the invention, compositions and methods are provided for prophylactically and/or therapeutically immunizing an individual against pathogens or abnormal disease-related cells. The vaccine can be any type of vaccine, such as a live attenuated vaccine, a cellular vaccine, a recombinant vaccine or a nucleic acid or DNA vaccine.

The present invention relates to compositions for delivering immunomodulatory proteins and methods of using the same.

Nucleic acid molecules can be delivered using one of several well known techniques including DNA injection (also referred to as DNA vaccination), recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vectors such as recombinant vaccinia.

DNA vaccines are incorporated herein by reference in U.S. Patents 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594 and incorporated herein by reference. Is described in the prioritized application. In addition to the delivery protocols described in this application, other methods of delivering DNA are described in US Pat. Nos. 4,945,050 and 5,036,006, which are incorporated herein by reference.

Routes of administration are topical, percutaneous, intramuscular, intranasal, intraperitoneal, epidermal, subcutaneous, intravenous, as well as mucosal tissues such as vaginal, rectal, intraurethral, buccal and sublingual tissues by inhalation or suppositories or washing. , Intraarterial, intraocular and oral. Preferred routes of administration include mucosal tissue, intramuscular, intraperitoneal, intradermal and subcutaneous injection. Genetic constructs may be administered by means including, but not limited to, conventional syringes, needleless injection devices, or “micro-propelled shock gene guns”.

When taken up by a cell, the genetic construct may remain in the cell as a functional extrachromosomal molecule and/or may integrate into the cell's chromosomal DNA. DNA can be introduced in the form of plasmids or plasmids, which remain as separate genetic material. In addition, linear DNA that can integrate into a chromosome can be introduced into cells. When introducing DNA into cells, reagents that promote DNA integration into chromosomes may be added. DNA sequences useful for facilitating integration can also be included in the DNA molecule. In addition, RNA can be administered to the cell. It is contemplated to provide the genetic construct as a linear small chromosome comprising centromere, telomere and origin of replication. The genetic construct may remain as part of the genetic material in a viable attenuated microorganism or a recombinant microbial vector that survives in a cell. The genetic construct may be part of the genome of a recombinant viral vaccine, where the genetic material integrates into the chromosome of the cell or remains extrachromosomal. The genetic construct contains the regulatory factors necessary for gene expression of the nucleic acid molecule. These factors include promoter, start codon, stop codon and polyadenylation signal. Additionally, enhancers are required for gene expression of sequences encoding target proteins or immunoregulatory proteins. These factors need to be operably linked to the sequence encoding the protein of interest and that the regulatory factor is operable in the individual to which they are administered.

The start and stop codons are generally considered to be part of the nucleotide sequence encoding the protein of interest. However, these factors need to be functional in the individual to which the genetic construct is administered. The start and end codons must be in frame with the coding sequence.

The promoter and polyadenylation signal used must be functional in the individual's cell.

Examples of promoters useful for carrying out the present invention, particularly in the production of human genetic vaccines, include Simian virus 40 (SV40), mouse mammalian tumor virus (MMTV) promoter, BIV long terminal repeat of human immunodeficiency virus (MV). (LTR) promoter, Moloney virus, ALV, CMV imatinate early promoter of cytomegalovirus (CMV), Epstein Barr virus (EBV), Raus sarcoma virus (RSV) promoter, as well as human actin, human myosin, human Promoters of human gene origin such as hemoglobin, human muscle creatine and human metallothionein.

Examples of polyadenylation signals useful for carrying out the present invention, particularly in the preparation of genetic vaccines for humans, include, but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV 40 polyadenylation signal (referred to as the SV40 polyadenylation signal) in the pCEP4 plasmid (Invitrogen, San Diego, CA) is used.

In addition to the regulatory factors required for DNA expression, other factors may also be included in the DNA molecule. These additional factors include enhancers. The enhancer may be selected from the group including, but not limited to, human actin, human myosin, human hemoglobin, human muscle creatine, and viral (CMV, RSV and EBV) enhancers.

The genetic construct is provided with a mammalian origin of replication so that the construct is maintained outside the chromosome and the construct of multiple copies can be generated within the cell. Plasmids pVAX1, pCEP4 and pREP4 (Invitrogen (San Diego, Calif.)) contain the origin of replication of Epstein Barr virus and the nuclear antigen EBNA-1 coding region that produces high copy number episomal replication without integration.

In some preferred embodiments relating to immunization applications, a nucleic acid molecule is delivered comprising a target protein, a nucleotide sequence encoding an immunomodulatory protein, and a gene for a protein that further enhances an immune response against the target protein. Examples of such genes include alpha-interferon, gamma-interferon, platelet-derived growth factor (PDGF), TNF, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and other cytokines such as IL-15 and lymphokines, such as IL-15, including IL-15 in which the signal sequence is deleted and optionally contains the signal sequence of IgE. If for any reason it is desired to remove the cells to which the genetic construct has been administered, additional factors can be added that act as targets for destroying the cells. The expressed form of the herpes thymidine kinase (tk) gene may be included in the gene construct. The drug gangcyclovir can be administered to an individual and the drug selectively kills any cells that produce tk, providing a means to selectively destroy cells using the genetic construct.

In order to maximize protein production, also suitable regulatory sequences can be selected for gene expression in the cells to which the construct is administered. Moreover, codons that are more efficiently transcribed in cells can be selected. One of skill in the art can prepare DNA constructs that are functional in cells.

One method of the present invention comprises mucosal tissue by washing selected from the group consisting of intramuscular, intranasal, intraperitoneal, subcutaneous, intradermal or topical or inhalation, vaginal, rectal, intraurethral, buccal and sublingual. And administering the nucleic acid molecule.

In some embodiments, the nucleic acid molecule is delivered to the cell in connection with the administration of a polynucleotide functional enhancer or gene vaccine promoter. Polynucleotide functional enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428 and International Application No. PCT/US94/0089 filed Jan. 26, 1994, which are incorporated herein by reference. Genetic vaccine promoters are described in U.S. Patent Application No. 021,579, filed April 1, 1994, which is incorporated herein by reference. An auxiliary agent administered in association with the nucleic acid molecule is administered as a mixture with the nucleic acid molecule or separately and simultaneously before or after administration of the nucleic acid molecule. Additionally, other agents that can function as transfection and/or replication and/or inflammatory agents and that can be administered concurrently with GVF include growth factors, cytokines and lymphokines, such as α-interferon, gamma-interferon. , GM-CSF, platelet-derived growth factor (PDGF), TNF, epidermal growth factor (EGF), ILA, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15, and As well as fibroblast growth factors, surface active agents such as immune stimulating complexes (ISCOMS), incomplete adjuvants of Fryund, LPS analogs including monophosphoryl lipid A (WL), vesicles such as muramyl peptides, quinone analogs and squalene, and Hyaluronic acid can also be used in conjunction with the administration of the genetic construct. In some embodiments, immunomodulatory proteins can be used as GVF. In some embodiments, the nucleic acid molecule is provided in association with the PLG to enhance delivery/uptake.

The pharmaceutical composition according to the present invention contains about 1 ng to about 2000 μg of DNA. In some preferred embodiments, the pharmaceutical composition according to the present invention comprises about 5 ng to about 1000 μg of DNA. In some preferred embodiments, the pharmaceutical composition contains about 10 ng to about 800 μg of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 μg of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 μg of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 μg of DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 μg of DNA.

The pharmaceutical composition according to the present invention is formulated according to the mode of administration used. When the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile in the absence of pyrogens and in the absence of particles. Preferably an isotonic formulation is used. In general, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizing agents include gelatin and albumin. In some embodiments, a vasoconstrictor agent is added to the agent.

According to some aspects of the invention, a method of inducing an immune response against an immunogen is provided by delivering a composition of the invention to a subject. The vaccine may be a live attenuated vaccine, a cellular vaccine, a recombinant vaccine or a nucleic acid or DNA vaccine.

In addition to using expressible forms that immunoregulate protein coding sequences to improve genetic vaccines, the present invention provides improved attenuated survival vaccines and improved using recombinant vectors to deliver foreign genes encoding antigens. It's about vaccines. Examples of attenuated survival vaccines and examples of vaccines using recombinant vectors that deliver foreign antigens can be found in US Pat. Nos. 4,722,848, incorporated herein by reference; 5,017,487; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,174,993; 5,223,424; No. 105,225,336; 5,240,703; 5,240,703; 5,242,829; 5,242,829; 5,294,441; 5,294,441; 5,294,548; 5,294,548; 5,310,668; 5,310,668; 5,387,744; 5,387,744; 5,389,368; 5,389,368; 5,424,065; 5,451,499; 5,451,499; 5,453,364; 5,462,734; 5,462,734; 5,470,734 and 5,482,713. Constructs are provided comprising a nucleotide sequence encoding an immunoregulatory protein and operably linked to a regulatory sequence that can act in a vaccine and be effective for expression. The genetic construct is incorporated into an attenuated survival vaccine and a recombinant vaccine to produce an improved vaccine according to the present invention.

The present invention relates to an improved method of immunizing an individual comprising the step of delivering the genetic construct to the individual's cells, provided with a portion of a vaccine composition comprising a DNA vaccine, an attenuated survival vaccine and a recombinant vaccine. . The genetic construct comprises a nucleotide sequence operably linked to a regulatory sequence that encodes an immunoregulatory protein and can be effective for expression by acting in a vaccine. The improved vaccine induces an enhanced cellular immune response.

Immunogen

The present invention is useful for inducing an enhanced immune response against a target protein, ie a protein that specifically associates with a pathogen, an allergen or an "abnormal" cell of the individual itself. The present invention is useful for immunizing individuals against pathogenic agents and bacteria such that an immune response against the pathogen protein provides protective immunity from the pathogen. The present invention is useful for eliminating hyperproliferative diseases and diseases such as cancer by inducing an immune response against a target protein that specifically associates with hyperproliferative cells. The present invention is useful for eliminating autoimmune diseases by inducing an immune response against a target protein that specifically associates with cells associated with autoimmune diseases.

According to some embodiments of the invention, DNA or RNA encoding a target protein and an immunomodulatory protein is introduced into tissue cells of an individual, where it is expressed to produce the encoded protein. The target protein, and DNA or RNA encoding one or both immunoregulatory proteins, are linked with regulatory factors required for expression in the cells of the individual. Regulatory factors for DNA expression include promoters and polyadenylation signals. In addition, other components, such as the Kozak region, may also be included in the genetic construct.

In some embodiments, a sequence in an expressible form encoding a target protein and a sequence in an expressible form encoding both an immunomodulatory protein have been found on the same nucleic acid molecule delivered to the individual.

In some embodiments, the sequence in the expressible form encoding the target protein occurs on a nucleic acid molecule separate from the nucleic acid molecule containing the sequence in the expressible form encoding one or more immunoregulatory proteins. In some embodiments, the sequence in the expressible form encoding the target protein and the sequence in the expressible form encoding the one or more immunoregulatory proteins are separate from the nucleic acid molecule containing the sequence in the expressible form encoding the one or more immunoregulatory proteins. It occurs on one nucleic acid molecule. A number of different nucleic acid molecules can be produced, delivered according to the invention, and delivered to individuals. For example, in some embodiments, the sequence in the expressible form encoding the target protein occurs on one nucleic acid molecule separate from the nucleic acid molecule containing the sequence in the expressible form encoding one or more immunoregulatory proteins. It occurs on a single nucleic acid molecule that is separate from the nucleic acid molecule containing the sequence in an expressible form that encodes the canine immunomodulatory protein. In this case, all three molecules are delivered to the individual.

The nucleic acid molecule(s) can be provided as plasmid DNA, which is the nucleic acid molecule of a recombinant vector, or as part of the genetic material provided in an attenuated or cellular vaccine. Alternatively, in some embodiments, the target protein and/or one or both immunomodulatory proteins may be provided as proteins in addition to or instead of the nucleic acid molecules encoding them.

The genetic construct may comprise a nucleotide sequence encoding a target protein or immunoregulatory protein operably linked to a regulatory factor required for gene expression. In accordance with the present invention, there is provided a combination of a genetic construct comprising a nucleotide sequence in an expressible form encoding a target protein and a nucleotide sequence in an expressible form encoding an immunoregulatory protein. Incorporation of DNA or RNA comprising a combination of genetic constructs into living cells results in the expression of the DNA or RNA and the production of a target protein and one or more immunoregulatory proteins. An enhanced immune response against the target protein is caused.

The present invention can be used to immunize individuals against all pathogens, such as viruses, prokaryotic and pathogenic eukaryotic bacteria, such as pathogenic unicellular bacteria and multicellular parasites. The present invention is particularly useful for immunizing individuals against pathogens that do not encapsulate while infecting cells, such as viruses, and prokaryotes such as gonorrhea, Listeria and Shigella. In addition, the present invention is also useful for immunizing individuals against protozoal pathogens, including the step of becoming intracellular pathogens in the life cycle. Table 1 provides a list of several viral classes and genera for which vaccines according to the present invention can be prepared. DNA constructs comprising a DNA sequence encoding a peptide comprising one or more epitopes that are identical or substantially similar to the epitopes appearing on a pathogen antigen, such as the antigens listed in the table, are useful in vaccines. In addition, the present invention is useful for immunizing individuals against multicellular parasites such as those listed in Table 2, as well as other pathogens, including prokaryotic and eukaryotic protozoal pathogens.

In order to prepare a genetic vaccine for prevention from pathogen infection, a genetic material encoding an immunogenic protein, from which a protective immune response can occur, must be included in the genetic construct as a coding sequence for the target. Regardless of whether the pathogen infects intracellularly (the present invention is particularly useful intracellularly) or extracellularly, it is unlikely that all pathogen antigens induce a protective response. Since both DNA and RNA are relatively small and can be prepared relatively easily, the present invention provides the additional advantage of being able to be vaccinated with multiple pathogen antigens. Genetic constructs used in genetic vaccines may contain genetic material encoding multiple pathogen antigens. For example, several viral genes can be included in a single construct, thereby providing multiple targets.

Tables 1 and 2 contain a list of several pathogenic agents and bacteria, and genetic vaccines for this can be prepared to protect individuals from infection by them. In some preferred embodiments, the method of immunizing an individual from a pathogen is against HIV, HSV, HCV, WNV or HBV.

Another method of the invention provides a method of providing a protective immune response against hyperproliferating cells, characterized by a hyperproliferative disease, and a method of treating an individual suffering from a hyperproliferative disease. Examples of hyperproliferative diseases include all forms of cancer and psoriasis.

It has been found to produce these proteins in cells of vaccinated individuals by introducing into the cells of the individual a genetic construct comprising a nucleotide sequence encoding an immunogenic “hyperproliferating cell”-associated protein. In order to immunize the hyperproliferative disease, a genetic construct comprising a nucleotide sequence encoding a protein associated with the hyperproliferative disease is administered to an individual.

In order to target the hyperproliferative-associated protein as an effective immunogenic target, it must be a protein produced only in hyperproliferative cells or at higher levels in hyperproliferative cells compared to normal cells. Target antigens include such proteins, fragments and peptides thereof; It includes one or more epitopes found on these proteins. In some cases, the hyperproliferative-associated protein is the product of a mutation of the gene encoding the protein. The mutated gene encodes a protein that is nearly identical to a normal protein, except that it has slightly different amino acid sequences that result in different epitopes not found on the normal protein. Such target proteins include proteins encoded by oncogenes such as myb, myc, fyn and translocation genes bcr/abl, ras, src, P53, neu, trk and EGRF. In addition to oncogene products as target antigens, target proteins for anticancer treatment and prophylaxis regimens are the variable regions of antibodies produced by B cell lymphoma, and in some cases T cells of T cell lymphoma, which are also used as target antigens for autoimmune diseases. Includes the variable part of the receptor. Other tumor-associated proteins can be used as proteins found at higher levels in tumor cells, for example proteins recognized by monoclonal antibody 17-IA and target proteins such as folic acid binding proteins or PSAs.

While the present invention is used to immunize individuals from one or more forms of cancer, the present invention is particularly useful for prophylactically immunizing individuals who are predisposed to developing certain cancers or have cancer and are therefore prone to recurrence. As well as in epidemiology, genetics and technology have developed, enabling the assessment of the probability and risk of cancer in individuals. Genetic screening and/or a family history of health can be used to predict the probability of developing any one in multiple types of cancer in a particular individual.

Similarly, individuals who have already developed cancer and those who have undergone cancer-removal treatment or who have otherwise palliated are particularly prone to recurrence and recurrence. As part of a treatment regimen, individuals can be immunized from cancer to prevent recurrence in individuals diagnosed with cancer. Thus, once an individual has one type of cancer and is known to be at risk of recurrence, they can be immunized to create their immune system that prevents the development of any future cancer.

The present invention provides a method of treating an individual suffering from a hyperproliferative disease. In this method, the introduction of the genetic construct acts as an immunotherapy to direct and stimulate the individual's immune system to eliminate hyperproliferative cells that produce the target protein.

The present invention provides methods of treating individuals suffering from autoimmune diseases and diseases by eliciting a wide range of protective immune responses against targets associated with autoimmunity, including cells that produce cellular receptors and "self"-directed antibodies. .

T cell mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (1DDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, Dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerative colitis. Each of these diseases is characterized by a T cell receptor that binds to an endogenous antigen and initiates an inflammatory chain reaction associated with an autoimmune disease. Vaccination against the variable regions of T cells eliminates these T cells by inducing an immune response, including CTLs.

In RA, it is characterized by a variable region of several specific T cell receptors (TCRs) associated with these diseases. These TCRs include Vβ-3, Vβ-14, 20 Vβ-17 and Vα-17. Thus, vaccination with DNA constructs encoding one or more of these proteins will elicit an immune response targeting RA-associated T cells [Howell, M.D., et al., 1991 Proc. Nat. Acad. Sci. USA 88: 10921-10925; Piliard, X., et al, 1991 Science 253: 325-329; Williams, W.V., et al., 1992 J Clin. Invest. 90: 326-333; Each of these is incorporated herein by reference]. In MS, several specific variants of the TCR associated with MS disease have been characterized. These TCRs include VfP and Va-10. Thus, vaccination with DNA constructs encoding one or more of these proteins will elicit an immune response targeting T cells associated with MS [Wucherpfennig, K.W., et al., 1990 Science 248: 1016-1019; Oksenberg, J. R., et al, 1990 Nature 345: 344-346; Each of these is incorporated herein by reference].

In scleroderma, several specific variables of the TCR associated with scleroderma have been characterized. These TCRs include Vβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. Thus, vaccination with DNA constructs encoding one or more of these proteins will elicit an immune response targeting T cells associated with scleroderma.

To treat patients suffering from T cell mediated autoimmune diseases, a synovial biopsy can be performed, particularly in patients whose variable regions of the TCR have also been characterized. Samples of the T cells present were obtained and the variable regions of these TCRs were identified using standard techniques. Genetic vaccines can be prepared using this information.

B cell mediated autoimmune diseases include lupus (SLE), hyperthyroidism, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosis and pernicious anemia. Each of these diseases is characterized by an antibody that binds to an endogenous antigen and initiates an inflammatory chain reaction associated with an autoimmune disease. Vaccination against the variable region of the antibody induces an immune response, including CTLs, thereby eliminating these B cells that produce the antibody.

In order to treat patients suffering from B cell mediated autoimmune diseases, it is necessary to identify the variable regions of the antibodies involved in autoimmune activity. A biopsy can be performed and a sample of the antibody present at the site of inflammation can be obtained. The variable regions of these antibodies can be identified using standard techniques. Genetic vaccines can be prepared using this information.

In the case of SLE, one antigen is believed to be DNA. Thus, in patients immunized against SLE, their sera can be screened for anti-DNA antibodies, and vaccines comprising DNA constructs encoding the variable regions of these anti-DNA antibodies found in serum can be prepared. have.

The typical structural features between the variable regions of both TCR and antibodies are well known. DNA sequences encoding specific TCRs or antibodies are generally as described in Kabat, et al., 1987 Sequence of Proteins of immnunological Interest US Department of Health and Human Services, Bethesda MD, incorporated herein by reference. It can be found according to a well-known method. In addition, general methods of cloning functional variable regions from antibodies are described in Chaudhary, VK, et al., 1990 Proc. Natl. Acad Sci. USA 87: 1066].

Recombinant protein production

The present invention relates to an in vitro host cell culture comprising an expression vector operative for host cells comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence; Such nucleic acid molecules; And to a host cell comprising such a vector. In addition, the present invention relates to a method of producing a fusion protein, including culturing a host cell. The present invention relates to an isolated fusion protein comprising an IgE signal peptide linked to a non-IgE protein sequence.

Fusion proteins can be prepared by conventional means using readily available starting materials as described above. By providing a suitable DNA sequence encoding the protein of interest, it is possible to prepare a protein using recombinant techniques known to date in the art.

Those skilled in the art can insert the DNA encoding the fusion protein into commercially available expression vectors for use in well known expression systems using well known techniques. Yeast S. A commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) can be used for production in the cerevisiae strain. For production in insect cells, a commercially available MaxBacTM (Invitrogen, San Diego, Calif.) complete baculovirus expression system can be used. Commercially available plasmid pcDNA I (Invitrogen, San Diego, Calif.) can be used for production in mammalian cells, such as Chinese hamster egg cells. Those skilled in the art can use these commercially available expression vector systems to prepare fusion proteins using conventional techniques and readily available starting materials.

Those skilled in the art can use other commercially available expression vectors and systems, or use well-known methods and readily available starting materials to prepare the vectors. Expression systems containing the necessary regulatory sequences, such as promoters and polyadenylation signals and preferably enhancers, are readily available and are known in the art for a number of hosts. See Sambrook et al. , Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)]. Thus, the protein of interest can be produced in both prokaryotic and eukaryotic systems, resulting in a range of processed forms of the protein.

In addition, a wide range of eukaryotic hosts are currently available for the production of recombinant heterologous proteins. Eukaryotic hosts can be transformed using expression vectors that directly produce the protein of interest using IgE signal peptides.

Commonly used eukaryotic systems include, but are not limited to, yeast, fungal cells, insect cells, mammalian cells, algal cells and higher plant cells. Suitable promoters, such as baculovirus polyhedral promoters, are available that are terminal sequences and enhancers, as well as compatible and operative for use in each of these host types. As above, promoters can be constitutive or inducible. For example, in mammalian systems, the mouse metallotionene promoter can be induced by adding heavy metal ions.

Matters for constructing an expression system suitable for the host of interest are known to those skilled in the art. After appropriately ligating the DNA encoding this into the selected expression vector, transforming a recipient-qualified host, and then culturing and maintaining under conditions in which heterologous gene expression occurs for recombinant production of the protein. Thus, the produced protein of the present invention is recovered from the culture by lysing the cells or preferably from a culture medium known and suitable in the art.

Those skilled in the art can use well-known techniques to separate the fusion receptor proteins and fragments thereof prepared using such an expression system.

The present invention can improve vaccines or immunological compositions and methods for prophylactically and therapeutically immunizing individuals against immunogens.

1 shows the data of Example 1 showing the production of IFN-γ after stimulation of human PBMCs with monoclonal antibodies against IL-15 and CD3. PBMCs were obtained from HIV-1 chronically infected subjects treated with triple therapy (HAART). The viral load of all donors is less than 500 copies/ml and their CD4 count is more than 500 cells/ml. To determine whether IL-15 enhances IFN-γ production as an indicator of effector function, these cells were stimulated with IL-15 and anti-CD3 and analyzed by standard ELI spot assay.
Figure 2 shows the data of Example 1 showing that the production of IFN-γ is mainly CD8 mediated after stimulation of monoclonal antibodies against IL-15 and CD3. As described in Figure 1, CD4 or CD8 T cells were removed from PBMCs from HIV-1 chronically infected subjects treated with triple therapy (HAART), followed by stimulation of the PBMCs with IL-15 and anti-CD3. Analyzed by standard ELI spot analysis.
3A, 3B, 3C and 3D show the data of Example 1 showing antigen specific production of IFN-γ after stimulation of human PBMCs with HIV-1 peptide and IL-15. PBMCs obtained from HIV-1 chronically infected subjects treated with triple therapy (HAART) were 25 ng/ml of IL-15 (Figures 3A and 3C) and HIV-1 Gag in combination with IL-15 in a standard ELI spot assay. They were analyzed for their ability to secrete IFN-γ in response to the peptides (Figures 3B and 3C). CD8 was removed and the production of IFN-γ was also evaluated after stimulation with HIV-1 peptide and IL-5 (FIG. 3D ).
Panels a, b and c of Figure 4 show the data of Example 1 showing HIV-1 antigen specific cellular immune response after immunization with HIV-1 DNA vaccine and IL-15. Balb/c mice were simultaneously injected with 50 µg of pCenv or pCgag or 50 µg of pIL-15 (IL-15 expression plasmid) at 0 and 2 weeks. Splenocytes were harvested 2 weeks after the final immunization. In panel a of Figure 4, splenocytes were tested by standard chromium release assay for CTL activity against HIV-1 enveloped and recombinant vaccinia infected P815 cells. In panel b of FIG. 4, the level of secretion of HIV-1 antigen-specific chemokines was analyzed. Splenocytes were stimulated with HIV-1 env recombinant vaccinia infected P815 cells. On the third day, the supernatant was collected and tested for secretion of MIP-1β. In panel c of FIG. 4, the level of antigen-specific secretion of IFN-gamma was evaluated. Splenocytes were resuspended at 5 x 10 ⁶ cells/ml. 100 μl of the titrant was added to each well of a 96-well microtiter flat bottom plate. Recombinant p24 protein is added to the wells 3 times to bring the final concentrations to 5 μg/ml and 1 μg/ml. The cells were incubated at 37° C. in _{5% CO 2} for 3 days, and the supernatant was collected. The level of secreted cytokines was measured using a commercially available ELISA kit.
Panels a and b of FIG. 5 depict the data of Example 1 showing intracellular staining for Th1 cytokines. Mice were injected with pCgag alone or pCgag and pIL-15 DNA plasmid twice. After 1 week, splenocytes were harvested and incubated in vitro for 5 hours in a medium containing p55 peptide pool (containing 127 15-mer spanning HIV-1 p55 with 11aa duplexes) and Brefeldin A. After stimulation, cells were stained extracellularly with anti-mouse CD3 and anti-mouse CD8 antibodies followed by intracellular staining with anti-mouse. Panel a of Figure 5 shows the data for IFN-γ. Panel b of FIG. 5 shows data for tumor necrosis factor-α. Dot plot shows response from CD3+/CD8+ lymphocytes.
6 shows the data of Example 1 regarding the murine T helper cell proliferation assay. Balb/c mice were simultaneously injected with 50 µg of pCgag or 50 µg of plasmid expressing the cDNA of pCenv and IL-2R-dependent Th1 cytokines IL-2 or IL-15 at weeks 0 and 2. One titrant of 100 μg containing 5 x 10 ⁵ cells was immediately added to each well of a 96 well microtiter flat bottom plate. Recombinant p24 protein was added to the wells 3 times so that the final concentrations were 5 μg g/ml and 1 μg g/ml. The stimulation index was measured. The spontaneous counting wells contain 10% fetal bovine serum used as an irrelevant protein control. Similarly, pCgag or control typically has an SI of 1 for their unrelated gp 120 protein. To confirm that the cells are healthy, PHA or Con A (Sigma) was used as a positive control for a polyclonal stimulator.
Figure 7 shows the data of Example 1 for epitope mapping of Gag in Balb/c mice after immunization with the DNA vaccine pCgag. Balb/c mice were simultaneously injected with 50 μg of pCgag and 50 μg of pIL-15 plasmid or gene IL-15 or vector skeleton expressing the vector skeleton at 0 and 2 weeks. Splenocytes were isolated and a standard ELI spot assay was set up using a series of peptides. The peptides were mixed in a series of 22 pools in matrix format and tested for their ability to activate cells to produce IFN-γ.
Panels a, b and c of FIG. 8 show the data of Example 1 showing the production of IFN-γ after stimulation of splenocytes derived from CD4 knockout mice. In panel a of FIG. 8, Balb/c mice were simultaneously injected with 50 μg of pCgag and 50 μg of pIL-15 (IL-15 expression plasmid) at 0 and 2 weeks. Splenocytes were harvested 2 weeks after the final immunization and tested for HIV-1 specific production of IFN-γ using an ELI spot. In panel b of Figure 8, Cd4 ^tm1Knw mice were immunized with pCgag with and without pIL-15. ^{Cd4 tm1Knw} mice in panel c of FIG. 8 were immunized with pCgag in combination with pIL-15, pCD40L, or both. Splenocytes were harvested 2 weeks after the final immunization and analyzed for HIV-1 Gag specific production of IFN-gamma after in vitro stimulation with HIV-1 Gag peptide.
Figure 9 shows the data of Example 2 showing that local production of IL-15 and CD40L at the vaccine site can replace the aid of T cells required for expansion of CD8 effector T cells.
Panels a to c of FIGS. 10, 11 and 12, panel ab of FIG. 13, and FIGS. 14 and 15 refer to the description presented in Example 3.
16 refers to the data presented in Example 4.

Example 1

Introduction

The success of anti-retroviral combination therapy to reduce the number of viruses in infected subjects has improved the prognosis in many HIV-1 positive individuals. However, many laboratories report that established viral pathogens are weakly affected by drug combination regimens (Refs. 1 to 3 below). To date, no combination therapy approach has resulted in viral clearance, and there are significant side effects associated with recent therapeutic regimens that ultimately affect patient compliance and affect disease progression. Therefore, the search for alternative forms of treatment, including effective immunotherapeutic approaches to HIV-1, is highly demanded. CD8+ T cell responses are thought to be important in controlling HIV-1 infection and slowing disease progression. The clear function of the HIV-1 specific CD8+ T cell response in regulating viral replication is not fully elucidated, but between long-term non-progressive and specific CD8+ T cell mediated cellular responses in individuals seropositive to HIV-1. The relevance of was established (Refs. 4 to 7 below). In addition, antibody responses were demonstrated by a cohort of individuals who were significantly exposed but HIV negative in Gambia, and showed anti-HIV-1 CD8+ T cell immune responses (Refs. 8 and 9 below). Indeed, after HIV-1 infection, a healthy cellular immune response is induced with simultaneous decrease in the number of viruses. However, despite the presence of high levels of HIV specific cytotoxic T lymphocytes (CTLs), HIV-1 infection is not eliminated. This discrepancy between a high CD8 mediated response and subsequent disease progression is of interest. The inability of CTLs to clear the virus is in part due to CTL escape mutations (references 10-14 below), potential viral immunopathologic mechanisms, e.g., Nef-related subregulation of MHC type I or Vpr on host immunity or It may be due to the Env effect (references 15 to 18 below). An additional problem is the lack of effective CD4+ T cell aid on CD8+ T lymphocytes (Refs. 19 and 20 below). It has been observed that circulating CD8+ cells may have impaired function (Ref. 21 below). Where the HIV-1 immunopathology limits the development of an effective CD8 response, the provision of the HIV-1 antigen in anti-retroviral therapy can increase CD8 memory cells and effector cells in a limited manner. This occurrence can have a strong effect on the disease process. However, it may be important to provide assistance for CD8+ T cell proliferation. In this regard, it has been found that the survival of CD8+ memory T cells is not dependent on continuous antigen presentation (Ref. 22 below), but may rather depend on the production of specific cytokines in the peripheral environment.

One such cytokine that appears to have a significant effect on CD8+ T cells is interleukin-15 (IL-15). Waldmann and his colleagues first reported that IL-15 is a 15 kDa protein that uses the gamma and beta chains of the IL-2 receptor complex with an alpha chain unique to signal T cells (Ref. 23 below). . IL-15 exhibits anti-apoptotic activity and appears to play a role in stimulating the memory CD8+ T cell phenotype. The role of IL-15 in HIV-1 infection is being studied by a number of groups. IL-15 has been demonstrated to reduce apoptosis of lymphocytes isolated from HIV-1 infected subjects (Ref. 24 below) and increase the activity and proliferation of natural killer cells (Refs. 25 to 27 below). In addition, IL-15 is involved in B cell proliferation (references 28 and 29 below) and activation of macrophages (reference 30 below) in HIV-1 infected subjects. Importantly, IL-15 also appears to play a direct role in HIV-1 effector T cell proliferation and interferon-gamma (IFN-γ) production (references 31 and 32 below). However, IL-15 was unable to stimulate IFN-γ in a number of subjects tested that were serologically positive for HIV-1. Thus, the effect of IL-15 on antigen specific CD8+ T cell immune responses was explored.

The effect of IL-15 on T cells isolated from chronically infected HIV-1 seropositive subjects was studied. It was found that rhIL-15 enhances the proliferation of CD8 T cells, and importantly, IL-15 increased effector antigen specific CD8+ IFN-γ production in all subjects. In the immunization model, IL-15 increased CD8+ effector function, which was explored in the immunization model system. CD8+ lymphocytes from mice were able to lyse targets expressing the HIV-1 antigen at higher levels when IL-15 was given in trans. This effect occurred in the absence of strong proliferation of CD4+ T cells. However, in CD4 knockout (KO) mice, IL-15 could not completely avoid the need for CD4 aid in the generation of a CD8 effector response. This suggests that although IL-15 is quite effective in proliferation of CD8 memory cells, IL-15 alone is not sufficient for their initial production.

Substance and method

ELI spot analysis on human PBMC

PBMCs isolated from HIV-1 positive volunteers by basic ficoll-hypaque technique were evaluated for effector function with standard ELI spot analysis. PBMC were resuspended at a concentration of ^{1 x 10 6} cells/ml in RPMI containing 10% FCS (R10). Antibody 1-DIK (Mabtech, Mariemont OH; Nacka, SE) was diluted to 15 μg/ml in 0.1 M carbonate-bicarbonate solution (pH 9.6), and 96-well nitrocellulose membrane plates (Millipore, Bedford, MA) were prepared. It was used to coat. Plates were incubated overnight at 4°C. The plate was washed 6 times with 200 μl of PBS. A mixture of 122 sterile peptides was prepared as a cocktail at a concentration of 50 μg/µl (for each peptide) in DMSO. The peptide is a series of overlapping peptides of 15 amino acids in length and includes all HIV-1 Gag (AIDS Reagent and Reference Repository, ARRR). 100,000 PBMCs were diluted 1:200 in R10 with or without 50 ng/ml of IL-15 in each well (100 μl @ 1.0 x 10 ^{6 cells/ml) of a nitrocellulose antibody-coated plate (} Final concentration 25 ng/ml) was added with 100 μl of peptide cocktail. Each sample was analyzed 3 times. PHA was used as a positive control at 5 μg/ml. The plate was incubated at 37° C. for approximately 24 hours. Then, the plate was washed 6 times with 200 μl of PBS. 100 μl of antibody 7-B6-1-biotin (Mabtech) was added to each well at a concentration of 1 μg/ml in PBS. Plates were incubated for 2-4 hours at room temperature. The plate was washed 6 times with 200 μl of PBS. 100 μl of streptavidin-ALP (Mabtech) was added to each well at a concentration of 1 μg/ml in PBS. Plates were incubated for 1-2 hours at room temperature. The plate was washed 6 times with 200 μl of PBS. 100 μl of substrate solution (BCIP/NBT, Sigma) was added to each well. The developer was removed with tap water. CD8 and CD4 populations were removed using Dynabeads (Dynal Biotech, Lake success, NY; Oslo, NO) coupled to monoclonal antibodies specific for either CD8 or CD4.

Co-stimulation of PBMC with monoclonal antibody against CD3

PBMCs isolated from subjects that were seropositive against HIV-1 were stimulated with a monoclonal antibody specific for CD3 bound to Dynabeads (Dynal Biotech) with or without IL-15 (50 ng/ml) and , The production of IFN-γ was analyzed by ELI spot as described above. CD8 and CD4 populations were removed using Dynabeads (Dynal Biotech) coupled to monoclonal antibodies specific for either CD8 or CD4.

Simultaneous stimulation of PBMC using CD40L

The CD40L protein was tested in combination with IL-15, and the peptide was mixed at a concentration of 250 μg/ml and the production of IFN-γ was analyzed by ELI spot as described above.

Plasmid Immunization in Mice

Female Balb/c mice were co-vaccinated with 50 μg of pCgag or pCenv at 0 to 2 weeks and 50 μg of plasmid expressing the gene of IL-2R-dependent Thl cytokine IL-15 as described above (see below. Document 33). Homozygous ^{mice for the} Cd4 tmIKnm targeted mutation were also used. These mice were completely blocked from development of ^{CD4 +} T-cells due to mutations in the CD4 gene; 90% of these circulating T-cells are CD8 ⁺ . Homozygous mutant mice also display type II restricted deletions in helper T-cell activity and other T-cell responses. B6.129S6-Cd4 ^tmIKnm was co-vaccinated with 50 μg of pCgag at 0 and 2 weeks, and 50 μg of plasmid expressing CD40L, IL-15, or both. All DNA was prepared using a Qiagen column, and the final formulation was 0.25% bupivacaine in isotonic citrate buffer. The spleen was harvested 1 week after the second injection.

Rat cytotoxic T lymphocyte analysis

^{The CTL response was evaluated by a 5 hour 51} Cr release CTL assay using recombinant vaccinia infected cells as targets. Splenocytes were isolated 1 week after vaccination and stimulated in vitro. Effectors were stimulated with related vaccinia-infected cells. P815 was infected with vDK1 for gag/pol and (ARRR) or vMN462 (ARRR) for env. The stimulant was fixed with 0.1% glutaraldehyde as described above, and incubated with splenocytes at a ratio of 1:20 for 4 to 5 days in CTL culture medium. The CTL culture medium is Hanks containing a 1:1 ratio of Iscove modified Dulbecco's medium (Gibc-BRL, Grand Island, NY) and 10% fetal bovine serum 1640 (Gibco-BRL). It consists of 10% RAT-T-STIM without equilibrium salt solution (Gibco-BRL) and Con A (Becton Dickinson Labware, Bedford, MA). Vaccinia-infected targets were prepared by infecting ^{3 x 10 6} P815 cells with a multiplicity of infection (MOI) of 10 for 12 hours at 37°C. A standard chromium release assay was performed, where the target cells _{were labeled with 20} ^{μCi/ml of Na 2 5l} CrO ₄ for 120 minutes and incubated with the stimulated effector splenocytes at 37° C. for 6 hours. CTL lysates were measured at an effector:target (E:T) ratio ranging from 50:1 to 12.5:1. The supernatant was collected and counted on an LKB CliniGamma gamma-counter. The specific dissolution rate is calculated by the following equation:

Maximum release was determined by lysis of target cells in medium containing 1% Triton X-100. If the value for the'spontaneous release' number exceeded 20% of the'maximum release', the analysis was not considered valid.

Complement lysate of CD8+ T cells

After treatment with an anti-CD8 monoclonal antibody (Pharmingen, San Diego, CA), CD8+ T cells were removed from splenocytes by incubating for 45 minutes at 37° C. as described above with rabbit complement (Sigma) (below Reference 33).

Rat T helper cell proliferation assay

Lymphocyte proliferation assay was used to evaluate the overall immunocompetence of lymphocytes and to detect antibody-specific dividing cells. Lymphocytes were harvested from the spleen, red blood cells were removed, and prepared by washing several times with the fresh medium described above (Ref. 34 below). The isolated cells were resuspended at a concentration of ^{5 x 10 6 cells/ml.} An aliquot of 100 μl containing 5×10 ⁵ cells was added directly to each well of a 96 well microtiter flat bottom plate. Recombinant p24 protein was added to the wells 3 times so that the final concentrations were 5 μg/ml and 1 μg/ml. Cells were incubated for 3 days at 37° C. under 5% CO _2. 1 μCi of tritiated thymidine was added to each well, and the cells were incubated at 37° C. for 12 to 18 hours. The plates were harvested and the incorporated amount of tritiumated thymidine was measured with a Beta plate reader (Wallac, Turku, Finland). The stimulation index was calculated by the following equation:

Stimulation Index (SI) = (experimental number/voluntary number)

Spontaneous water wells contain 10% fetal bovine serum, which will serve as an unrelated protein control. Similarly, splenocytes from pCgag or control immunized mice generally have a value of SI 1 for their unrelated protein targets. To confirm that the cells were healthy, PHA or Con A (Sigma) was used as a polyclonal stimulant positive control.

Cytokine and chemokine analysis of stimulated mouse cells

Lymphocytes were harvested from the spleen, and the isolated cells were resuspended at a concentration of ^{5 x 10 6 cells/ml.} An aliquot of 100 μl containing 5×10 ⁵ cells was added to each well of a 96 well microtiter flat bottom plate. Recombinant p24 or envelope protein was added to the wells 3 times so that the final concentrations were 5 μg/ml and 1 μg/ml. The cells were cultured at 37° C. under 5% CO ₂ for 3 days, and the supernatant was collected. Cytokines and chemokines were measured using a commercially available ELISA kit.

Intracellular staining for interferon-γ in stimulated murine cells

Mice were given two injections containing pIL-15 with either pCgag DNA or pCgag DNA plasmid. One week later, splenocytes were incubated in vitro for 5 hours in a medium containing p55 peptide cocktail (containing 122 15-mer spanning HIV-1 p55 with 11aa duplex) and Brefeldin (Brefeldin)A. After stimulation, cells were stained extracellularly with anti-mouse CD3 and anti-mouse CD8 antibodies, followed by intracellular staining with anti-mouse IFN-γ. Dot plots represent responses from CD3+/CD8+ lymphocytes.

Epitope mapping

Splenocytes were resuspended at a concentration of ^{1 x 10 6} cells/ml in RPMI containing 10% FCS (R10). A series of 122 peptides obtained from the AIDS Reference and Reagent Repository were mixed as a pool of 10 peptides per pool at a final concentration of 20 μg/ml/peptide. Each peptide was included in two separate pools for a total of 22 peptide pools. The pools were arranged in a matrix format and used for splenocyte stimulation. IFN-γ production was evaluated as an ELI spot (R and D system). The plate was incubated at 37° C. for approximately 24 hours. Each sample was analyzed 3 times.

result

Stimulation of lymphocytes with CD3 and IL-15

For IL-15, its ability to increase T cell effector activation in a synergistic manner upon T cell receptor stimulation was evaluated. PBMCs were isolated from HIV-1 infected individuals. PBMC were stimulated with an antibody surface-bound to CD3, and then incubated with IL-15 overnight. As expected, CD3 stimulation of PBMC alone induced the production of IFN-γ, whereas IL-15 supplement alone induced low to no response. However, when lymphocytes were stimulated with CD3 and IL-15 together, it was observed that the number of cells secreting IFN-γ increased several times (FIG. 1). After removal of CD4+ or CD8+ T cells from the stimulated population, they were supplemented with IL-15 and tested for activity again. Again, the loss of CD8 cells eliminated the activation signal. These data indicate that CD8+ effector T cells from chronically infected HIV-1 individuals can be increased by IL-15/CD3 stimulation (FIG. 2 ).

Generation of antigen-specific IFN-7 in HIV-1 positive samples after IL-15 stimulation

The ability of IL-15 to enhance the HIV-1 antigen specific CD8+ response was evaluated in vitro. Samples were collected from chronically infected HIV-1+ subjects being treated with recombinant anti-retroviral therapy (HAART). For PBMCs, after stimulation with an HIV-1 specific peptide in the presence or absence of IL-15, it was evaluated for its ability to secrete IFN-γ. PBMCS were stimulated with overlapping HIV-1 amino acid peptides covering the entire open reading frame of the HIV-1 gag protein. PBMC from the subject stimulated with the peptide showed increased IFN-γ production when treated with IL-15 (Figs. 3A and 3B), and IFN-γ production with and without IL-15 There was a significant difference between them (p=0.009) (Fig. 3c). Some subjects had high levels of IFN-γ secretion when stimulated with IL-15 alone (Fig. 3A), suggesting that they have blocked partial T cell activation and require effective cytokine supplementation. When the CD8 cell population was removed, its activity was completely CD8 mediated as IFN-γ production was lost (Fig. 3D).

IL-15 enhances the CD8+ CTL response in mice in vivo.

By this study of the HIV-1 response and IL-15, it was established that IL-15 is capable of enhancing IFN-γ production in primed T cell populations. However, it was unclear what effect IL-15 has on the functional induction of CD8+ T cells in vivo. To solve this question, a mouse model system was used. Mice were vaccinated with the HIV-1 plasmid in such a way as to study the induction of CD8 immunity in vivo by delivering the HIV-1 antigen. The plasmid expressing HIV-1 was co-injected with either the plasmid expressing IL-15 or the control plasmid, and the obtained immune responses were compared. In volumetric CTL analysis, co-injection with the HIV-1 envelope and the plasmid expressing IL-15 was compared to the 11% lysis observed with the envelope plasmid and the control vector, with an effector:target ratio of HIV of 50:1. Caused nearly 40% dissolution of the -1 envelope-expressing target (FIG. 4, panel a). These results were CD8 T cell dependent, indicating a significant effect of IL-15 on effector T cell responses.

IL-15 induces secretion of MIP-lβ and IFN-g in mice after antigen stimulation.

The vaccine-induced cellular immune response was further evaluated by examining the expression profile of β-chemokine MIP-1β as an immune activation marker. Chemokines are important modulators of immune and inflammatory responses. They are particularly important for the molecular regulation of white blood cell trafficking from blood vessels to the peripheral sites of host defense. In addition, it has already been reported that T cell-generated chemokines, including MIP-1β, play a critical role in cellular immune proliferation (Ref. 24 below). Thus, chemokine levels produced by stimulated T cells can provide additional insight into the level and quality of antigen-specific cellular immune responses. The supernatant from the stimulated T cells (as described in Materials and Methods) was analyzed and tested for MIP-1β release. Co-immunization with IL-15 resulted in high levels of MIP-1β secretion (Figure 4, panel b).

In addition, the supernatant was also evaluated for the generation of Th1 cytokine and IFN-γ. After 3 days of stimulation of lymphocytes with stimulating cells infected with recombinant vaccinia expressing the HIV-1 envelope, samples were obtained immediately before the cells were used for CTL analysis. Figure 4, panel c, shows that splenocytes from mice co-injected with IL-15 induced higher levels of IFN-γ (120 pg/ml) compared to those injected with the plasmid vaccine alone or as a control. I'm doing it. In contrast, no significant production of IL-4 by any culture was observed in these studies (data not shown).

Intracellular staining for IFN-γ and TNF-α.

In order to quantify the T cell response to the HIV-1 vaccine, an intracellular cytokine staining assay was performed. The immunized animals were sacrificed, and splenocytes were harvested and cultured in vitro for 5 hours in a medium containing p55 cocktail mixture and Brefeldin A. CD8+ CD3+ T cells were analyzed for IFN-γ or TNF-α production by flow cytometry (FIG. 5, panel a and FIG. 5, panel b). IL-15 co-vaccinated animals exhibited a high CD8 effector T cell response, with 2.6% of CD8+ T cells producing IFN-γ and 3.7% producing TNF-α. These data exemplify that IL-15 exerts a significant effect on functional CD8+ T cell responses.

Lymphocyte proliferation of murine splenocytes co-immunized with IL-15 and HIV-1 vaccines.

Activation and proliferation of T helper lymphocytes is essential for increased humoral and cellular immunity. Splenocytes from immunized mice were evaluated by a basic lymphocyte proliferation assay for their ability to proliferate in response to stimulation of the recombinant HIV-1 antigen. IL-15 did not appear to have a significant effect on the proliferative response (FIG. 6 ). However, IL-2 was used as a control, and a significant increase in splenocyte proliferation for the gpl20 env protein was clearly observed in mice co-injected with the IL-12 plasmid. The splenocytes of mice co-injected with IL-12 caused a stimulation index that was about 3 times greater than that of the control, pCgag alone, or splenocytes of mice immunized with pCEnv + IL-15 (FIG. 6 ). These data appear that IL-15 enhances CD8 T cell function without a significant increase in T cell aid. In addition, this exemplifies that their augmentation by IL-15 is not dependent on IL-2. This suggests that in this case, CD4 as well as CD8 effector function is increased.

Epitope mapping

To address the question of whether the enhancement of IL-15-treated CD8+ T cell responses is due to an increase in the number of responded epitopes (i.e., epitope spread), or due to an overall increase in the number of CD8+ T cells specific to the same epitope. , ELI spot analysis and a series of peptides obtained from the AIDS Reference and Reagent Repository (mixed as a pool in matrix format) were used. Two epitopes were identified. Dominant epitopes were mapped to Gag amino acids 197 to 211 (AMQMLKETMEEAAE-SEQ ID NO: 1) (Figure 7). Paterson et al. recombined L. After immunization with the L. monocytogenes HIV-1 vaccine, AMQMLKETI-SEQ ID NO: 2 (reference 35 below) was already defined as the dominant CD8 epitope. The sub-dominant epitope, Gag amino acids 293 to 307 (FRDVDRFYKTRAE-SEQ ID NO: 3) (FIG. 7) was further defined. There was no increase in the number of epitopes reacted as in the response to both epitopes observed in the Gag-only group. However, IL-15 significantly increased the range of responses to these epitopes. The sub-dominant epitope was clearly demonstrated only in IL-15 co-vaccinated animals. IL-15 is effective in increasing effector CD8 cells.

CD4 knockout mouse

We observed that IL-15 increased antigen-specific CD8 T cells in PBMCs from HIV-1 infected individuals. In addition, we observed significant CD8 effector cell induction dependent on CD4 increase in this vaccine model. Thus, the contribution of CD4 helper cells to increased IL-15 immunity was questionable. To solve this problem, the ability of IL-15 to induce a population of CD8 effectors in the complete absence of CD4 cells was tested. Mouse homozygotes (ref. 36 below) were immunized against the Cd4 ^{tmIKmw targeted mutation.} Blocked CD4+ T-cell development in these mice, and therefore, most of the circulating lymphocytes are CD8 cells. On average, in a plasmid co-immunization model in which cells producing approximately 200 IFN-γ per million lymphocytes in normal mice are induced, in the complete absence of CD4 cells, IL-15 is able to rescue the induced CD8 effector function. None (Fig. 8, panel b). Since the effect of IL-15 does not appear to be associated with an increase in CD4 (Figure 6), this drawback is due to a lack of another function provided by T helper cells, and CD4 T helper cells also activate antigen presenting cells (APCs). To help increase CD8. In this model of APC activation, linking CD40 with T cell CD40 ligand on APC upregulates B7 expression, which allows T cell activation. The B7 molecule provides co-stimulation for CD8 T cell growth in the provision of MHC type I peptides. In addition, Bourgeois et al. (Ref. 37 below) demonstrated that CD40L can have a direct effect on the development of CD8 memory cells.

It was then considered and explored that the defects in CD4 aid clarify themselves to the level of defects in co-stimulation. To test this hypothesis, mice were co-immunized with pCgag and a plasmid containing both IL-15 and CD40L. An immobilized CD40L molecule was used. Immobilized CD40L is expressed locally, but not secreted in immune cell trafficking, which complicates the experiment (Ref. 38 below). Such vaccination can provide trans costimulation in a plasmid model (Ref. 38 below). Indeed, when pCgag was studied in combination with pCD40L, a Gag-specific CD8 immune response was induced in CD40 KO mice (FIG. 8). These data further indicate that IL-15 has a direct effect on memory CD8 lymphocytes. In the absence of CD4 cells, IL-15 is unable to induce antigen specific CD8 cell responses from non-sensitized cells.

Argument

Maintaining and enhancing the HIV-1 specific CD8 immune response has been the basis for a number of studies. In recent studies, it has been reported that IL-15 may play an important role in supporting memory cell survival. In a mouse model, it has been observed that the presence of IL-15 can cause memory cell division (Ref. 39 below). In studies with transgenic mice genetically deficient in IL12, IL-15 or their specific receptors, as well as in vitro functional assays were important to characterize the role by IL-15. Indeed, Zgang et al. (Ref. 39 below) demonstrated that in an in vivo mouse model, IL-15 provides an effective and differential stimulation of CD44hi CD8+ T cells, a memory phenotype. In addition, Ku et al. (Ref. 40 below) reported that the division of memory CD8+ T cells is stimulated by IL-15, but is suppressed by IL-2. In addition, it was found that IL-2 inhibited the proliferation of CD8+ memory T cells.

The literature described herein demonstrates that IL-15 is also particularly effective in inducing CD8+ effector T cells in vitro and in vivo. CD8+ T cells isolated from HIV-1 infected patients were able to secrete IFN-γ in an antigen-specific manner when incubated with the peptide and IL-15. IL-15 works with TCR to stimulate lymphocytes to produce IFN-γ, and exhibits an effector phenotype. In some subjects, IL-15 caused the production of IFN-γ in the absence of antigen. This indicates that in HIV infection, some cells are partially activated, and these partially activated states can be rescued by IL-15. However, it is important that in all subjects, when PBMC are stimulated with both IL-15 and HIV-1 antigens, there is a significant increase in effector function.

Recently, von Adrian and his colleagues (Ref. 41 below) suggested that IL-15 stimulation of lymphocytes can lead to CD8+ T cells that develop memory cell phenotypes directly from non-sensitized cells. have. However, our data suggests that the age of TCR may lead to more complete activation of CD8+ T lymphocytes, indicating that the effect of IL-15 alone on non-sensitized cells will be minimal. In addition, it has been suggested that IL-15 causes non-functional memory cells (Ref. 41 below). The data herein demonstrate that increasing IL-15 results in fully functional CD8+ T cells as assessed in humans as well as in mouse studies. In mice, IL-15 significantly increases the enhancement of the β-chemokine and IFN-γ responses as well as the CD8+ T cell response, suggesting an antigen-specific increase, which constitutes prior literature (Ref. 33 below. And 42). This increase in CD8+ T cell function was observed in the absence of CD4+ T cell increase. Additionally, CD4 T cells play an important role in the development of the CD8 response. In studies in CD4 knockout mice, the need for CD4 T cells could be overcome by using CD40L. These findings can be critically important in immunotherapy for viral infections.

Many immunotherapeutic strategies have focused on increasing CD8+ T cell responses. HIV-1 infection exacerbates immunotherapy through virus-induced immune suppression that contributes to a lack of effective CD4+ T cell aid. In addition, this lack of assistance appears to be responsible for non-productive CD8+ T cell responses. In general, chronic infections require CD4+ assistance to maintain viral replication control, similar to the case for HIV-1 infection. Serbina et al. (Ref. 43 below) demonstrated that CD8+ cytotoxic T cells are CD4+ T cell dependent. They further found that in CD4 T cell knockout mice, IL-15 production was reduced. However, IL-15 is not produced by CD4+ T cells. It is produced predominantly by stromal cells, monocytes and macrophages. There may be several feedback mechanisms by which CD4+ T cells enhance the production of IL-15, and in the case of reduced CD4 help, CD8+ T cell function is eventually reduced. With this feedback mechanism, it is possible to explain the cause of IFN-γ production in 3 out of 6 subjects after IL-15 alone was added. In the absence of CD4, and thus at lower levels of IL-15, residual virus can only partially activate CD8+ T lymphocytes in subjects that are seropositive to HIV-1. Importantly, it appears that the addition of IL-15 in trans could replace the deficiencies caused by viral immunosuppression. Implications from this hypothesis should be considered in the area of immunotherapy against HIV-1.

In summary, IL-15 increases CD8+ T cell effector function in mice and increases the functionality of CD8+ T cells isolated from subjects that are positive for HIV-1 infection. The use of IL-15 as a supplement to active immunotherapy should be considered as an adjuvant therapy to HAART.

References (each cited as a reference to the original)

Example 2

Production of CD4 (+) Th cells and IFN-γ is required to modulate viral replication in immunocompromised individuals as well as in anti-tumor immunology. Data from the experiments performed demonstrate that the need for T cells to aid in the expression of CD8 effectors or T cells can be replaced by local production of IL-15 and CD40L at the vaccine site. From experiments using mice in which CD4(+) T cells were removed by gene knockout of MHC II type beta chain (MHC II KO), priming of animals with antigen gag + IL-15 + CD40L resulted in activation of CD8 T cells. It has been found to cause. Activation is measured by IFN-γ production as a point. A score of 50 or more in this analysis is positive. These data, as shown in Table 9, illustrate a simple method for activation of CD8 T cells independent of effect or CD4 (+) T cell aid. These studies are important for the treatment of immunocompromised individuals.

Example 3

Human, mouse and ape IL-15cDNA encodes a 162 amino acid (aa) residue precursor protein containing a 48 aa residue leader, which is cleaved to yield 114 aa residue mature IL-15. Human IL-15 has approximately 97% and 73% identical sequences to ape and mouse IL-15, respectively. Both human and ape IL-15 are active on mouse cells. Although the structure of IL-15 has not been determined, it is predicted to be similar to IL-2 and other members of the cytokine family of quadruple-helix bundle structures [Grabstein, K. et al. (1994) Science 264: 965, Anderson, D. M. et al. (1995) Genomics 25:701; and Bamford, R. N. et al. (1995) cytokine 7: 595, Brandhuber, B. J. et al. (1987) Science 238: 1707, both of which are incorporated herein by reference].

IL-15 mRNA was detected in heart, lung, liver, placenta, skeletal muscle, adherent peripheral blood monocytes, APC (dendritic cells), and epithelial and fibroblast cell lines. However, IL-15 mRNA cannot be detected in activated peripheral blood T cells and contains high levels of IL-2 mRNA. IL-15 has been shown to stimulate the proliferation of natural killer cells, activated peripheral blood T lymphocytes, tumor infiltrating lymphocytes (TIL) and B cells. In addition, IL-15 has been shown to induce lymphokine-activated killer (LAK) activity in NK cells as a chemoattractant for human blood T lymphocytes and induce the production of cytolytic effector cells [Armitage, R.J. et al. (1995) J. Immunol. 154:483; P. Wilkinson and F. Liew (1995) J. Exp. Med. 181:1255; Grabstein, K. et al. (1994) Science 264:965; Giri, J.G. et al. (1994) EMBO J. 13:2822; and Giri, J.G. et al. (1995) EMBO J. 15:3654, each of which is incorporated herein by reference].

Because IL-15 is a prototype Thl cytokine and its activity as a stimulator of T cells, NK cells, LAK cells and TILs, IL-15 is a molecular adjuvant with DNA vaccines such as HIV vaccines that enhance cellular immune responses. It is an interesting candidate to use as. IL-15 increases HIV-specific CTL, and overproduction of IL-15 is associated with inflammatory diseases such as Crohn's disease.

Northern blot analysis shows widespread homeostatic expression of IL-15. Control of expression occurs at the level of translation and translocation (intracellular trafficking) after transcription. IL-15 mRNA contains a number of factors that interfere with its transcription to proteins including: 1) 5'AUG (5 of mice, human) loaded with upstream AUGs that interfere with effective IL-15 translation. Of 12); 2) a start codon for the IL-15 coding sequence, with a weak Kozak environment (GTAATGA); And 3) the presence of a negative factor in the C-terminus of the IL-15 mature protein coding sequence (Grabstein et al., (1994) Science 264: 965-968, Bamford et al., (1996) PNAS 93: 2897- 2902; Bamford et al., (1998) J. Immunol 160:4418-4426; and Kozak et al., (1991) J. Cell Biol. 115: 887-903, which is incorporated herein by reference]. Each of these three controls can be removed to enhance expression.

The original IL-15 isoform contained two leader peptides: 21 aa signal peptide (SSP) or 48 aa signal peptide (LSP) (Waldmann et al. Ann. Rev> Immunol. (1999) 17: 19-49, which is incorporated herein by reference].

The following strategy to increase the expression of IL-15 by optimizing the IL-15 DNA construct for immunization was followed. Primers are designed to amplify IL-15 from the start of the signal peptide, so the upstream inhibitory AUG is not present in the final IL-15 message. Primers were designed to contain a strong Kozak environment (GCCGCCACC). The C-terminal negative regulatory factor was removed using a PCR antisense primer design. Primers are shown in Figure 10.

The following strategy to increase IL-15 expression through substitution of the 48 amino acid IL-15 signal peptide (LSP) containing an IgE leader was performed. Sense primers were designed to start after 48 aa LSP while amplifying the antisense primer from the termination site. Primers were designed to contain a strong Kozak environment (GCCGCCACC-SEQ ID NO: 4). Sense primers were designed to contain the sequence for the IgE leader sequence + ATG start site. Primers are shown in Figure 11.

Various constructs were prepared and used to transfect RD cells. Measurements were made for various constructs for IL-15 protein production. Data are presented in Figure 12, panels a-c. 13A is a human construct comprising a coding sequence for a 21 amino acid signal peptide linked to IL-15 (IL-15SSP-left) and a coding sequence for a human 48 amino acid signal peptide (IL-15 LSP-right). The comparison of expression by is shown. Figure 12, panel b is by a human construct comprising the coding sequence for the 48 amino acid signal peptide (human IL-15 LSP-left) and the coding sequence for the IgE signal peptide (human IL-15-IgE-right). The comparison of expression is shown. 12, panel c is a macaque construct comprising a coding sequence for a 48 amino acid signal peptide (Mac IL-15 LSP-left) and a coding sequence for an IgE signal peptide (Mac IL-15-IgE-right). The comparison of expression by is shown.

The IL-15 bioactivity of the IL-15 protein produced from various constructs was measured. The data are shown in Figure 13 panels a and b. Figure 13, Panel a shows a human construct comprising a 48 amino acid signal peptide (human IL-15 LSP-left) and a human construct comprising a coding sequence for IgE signal peptide (human IL-15-IgE-right) A comparison of the IL-15 bioactivity between is shown. Figure 13, panel b shows a macaque construct comprising a coding sequence for a 48 amino acid signal peptide (Mac IL-15 LSP-left) and a macaque construct comprising a coding sequence for an IgE signal peptide (Mac IL-15-IgE-right) for IL-15 bioactivity.

Constructs were prepared using the expression vector pVAX by inserting the IL-15 coding sequence linked to the coding sequence for the IgE signal peptide. Constructs encoding HIV-1 Gag were also generated. Immunological experiments were performed to compare the effects on the immune response using IL-15 engineered plasmids combined with HIV-1 Gag. Balb/c mice were vaccinated according to the immunization schedule shown in Figure 14.

The immune response was studied by comparing the restimulation of antigen-specific IFN-γ production at 5 weeks after the third immunization. The data are presented in Figure 15. The vaccine group included non-sensitized mice, mice vaccinated with the vector pCDN3, mice vaccinated with a construct encoding HIV-1 Gag, a construct encoding HIV-1 Gag and IL-15 linked to a 48 amino acid signal peptide. Mice vaccinated with and vaccinated with a construct encoding HIV-1 Gag linked to an IgE signal peptide were included.

Example 4

The engineered IL-15 plasmid vaccine was constructed by removing the original IL-15 Kozak region, AUG and UTR. Engineered IL-15 plasmids were provided with the coding sequence for the IgE signal peptide. The engineered IL-15 was expressed at a level of 30 to 50 times higher than observed, comparable to that of the wild-type plasmid. The immune response observed in mice co-immunized with the processed IgE signal-IL-15 and HIV-1 gag constructs was a significant fold than in mice immunized with the HIV-1 gag construct alone. The data are presented in Figure 16.

Example 5

The isolated cDNA encoding the immunoregulatory protein is useful as a starting material for the construction of a construct capable of producing the immunoregulatory protein. In some embodiments, a construct is provided in which the coding sequence for one of the following immunomodulatory proteins is linked to an IgE signal peptide. In some embodiments, such constructs are provided as part of vaccines and immunomodulatory compositions such as those described herein.

Using standard techniques and readily available starting materials, nucleic acid molecules encoding immunomodulatory proteins can be prepared and incorporated into the constructs, vectors and vaccines described herein.

Genbank accession number AF031167 represents the complete coding sequence of human IL-15 mRNA. Genbank accession numbers Y09908, X91233, X94223 and X94222 also represent human IL-15 sequences. Each sequence is incorporated herein by reference.

Genbank accession number L07414 represents the complete coding sequence of human CD40-ligand mRNA. These sequences are incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for Bax is L22473, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for TRAIL is U37518 or AF023849, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for TRAILrecDRC5 is U90875 or AF016266, which is incorporated herein by reference. Also, TRAIL-R2 AF016849; TRAIL-R3 AF014794; And TRAIL-R4 AF021232 are incorporated by reference.

The Genbank accession number for the nucleotide and amino acid sequence for RANK is AF018253, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for the RANK ligand is AF019047 or AF333234, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for Ox40 is X75962, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for the Ox40 ligand is X79929 or AB007839, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for NKG2D is AF46181l or X54870, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for MICA is X92841, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for MICB is U65416, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for NKG2A is X54867, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for NKG2B is X54868, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for NKG2C is X54869 or AjO016984, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for NKG2E is L14542, which is incorporated herein by reference.

The Genbank accession number for the nucleotide and amino acid sequence for NKG2F is AH006173, U96845 or U96846, which is incorporated herein by reference.

Genbank accession number for the nucleotide and amino acid sequence for CD30 is M83554 [Durkop, H et al. Cell 68 (3), 421-427 (1992)], which is incorporated herein by reference.

Genbank accession number for the nucleotide and amino acid sequence for CD153 (CD30L) is L09753 [Smith, C. A., et al. Cell 73 (7), 1349-1360 (1993)], which is incorporated herein by reference.

Genbank accession numbers for the nucleotide sequence for Fos are K00650 or V01512, each of which is incorporated herein by reference.

Genbank accession numbers for the nucleotide sequence for c-jun are J04111 or M29039, each of which is incorporated herein by reference.

Genbank accession numbers for the nucleotide sequence for Sp-1 are BC021101, BC005250, BC002878, M31126, J02893 or X15102, each of which is incorporated herein by reference.

The nucleotide sequence for Apl is described in Lee et al, 1987 Cell 49: 741-752, Rauscher et al. 1988 Science 240: 1010-1016, and Chiu et al, 1988 Cell 54: 541-552, each of which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for Ap-2 is M36711, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for p38 is U66243, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for p65Rel is L19067, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for MyD88 is U70451, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for IRAK is NM001569, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for TRAF6 is U78798, which is incorporated herein by reference.

The nucleotide sequence for IkB is described in Gilmore et al. Trends Genet 1993 Dec; 9(12): 427-33, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for NIK is Y10256, which is incorporated herein by reference.

The nucleotide sequence for SAP K is described in Franklin et al. Oncogene. 1995 Dec 7; 11(11): 2365-74, which is incorporated herein by reference.

Genbank accession numbers for the nucleotide sequence for SAP1 are M85164 or M85165, each of which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK2 is L31951, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK1B2 is U35005, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK1B1 is U35004, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK2B2 is U35003, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK2B1 is U35002, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK1A2 is U34822, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK2A1 is U34821, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK3A1 is U34820, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for JNK3A2 is U34819, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for the NF-kappa-B2, p49 splicing form is A57034, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for the NF-kappa-B2, p100 splicing form is A42024, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for the NF-kappa-B2, p105 splicing form is S17233, which is incorporated herein by reference.

The Genbank accession number for the nucleotide sequence for the NF-kappa-B 50K chain precursor is A37867, which is incorporated herein by reference.

The nucleotide sequence for NFkB p50 is described in Meyer R., et al. (1991) Proc. Natl. Acad. Sci. USA 88(3), 966 970, which is incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-1 a are well known and are described in Telford, et al. (1986) Nucl. Acids Res. 14: 9955-9963, Furutani, et al. (1985) Nucl. Acids Res. 14: 3167-3179, March, et al. (1985) Nature 315: 641-647, and accession code Swissprot P01583, each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-2 are well known and described in Holbrook, et al. (1984) Proc. Natl. Acad. Sci. USA 81: 1634-1638, Fujita, et al. (1983) Proc. Natl. Acad. Sci. USA 80: 7437-7441, Fuse, et al. (1984) Nucl. Acids Res. 12: 9323-9331, Taniguchi, et al. (1983) nature 302: 305-310, Meada, et al. (1983) Biochem. Biophys. Res. Comm. 115: 1040-1047, Devos, et al. (1983) Nucl. Acids Res. 11: 4307-4323 and accession number Swissprot P01585, each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-4 are well known and are described in Arai, et al. (1989) J. Immunol. 142: 274-282 Otsuka, et al. (1987) Nucl. Acids Res. 15: 333-344, Yokota, et al. (1986) Proc. Natl. Acad. Sci USA 83: 5894-5898, Noma, et al. (1984) Nature 319: 640-646, Lee, et al. (1986) Proc. Natl. Acad. Sci. USA 83: 2061-2063, and the accession code Swissprot 05112 (the accession code for murine IL-4 is Swissprot 07750), each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-5 are well known and are described in Campbell, et al. (1987) Proc. Natl. Acad. Sci. USA 84: 6629-6633, Tanabe, et al. (1987) J. Biol. Chem. 262: 16580-16584, Campbell, et al. (1988) Eur. J. Biochem. 174: 345-352, Azuma, et al. (1986) Nucl. Acids Res. 14: 9149-9158, Yokota, et al. (1986) Proc. Natl. Acad. Sci. USA 84: 7388-7392, and accession code Swissprot P05113, each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-10 are well known and are described in Viera, et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1172-1176, and accession code Swissprot P22301.

The nucleotide and amino acid sequences of human IL-15 are well known and are described in Grabstein, et. al. (1994) Science 264:965-968, and accession code Swissprot U03099, each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human IL-18 are well known and are described in Ushio, et al. (1996) J. Immunol. 156: 4274-4279, and accession code D49950, each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human TNF-α are well known and are presented in Pennica, (1984) Nature 312: 724-729, and accession code Swissprot P01375, each of which is incorporated herein by reference.

The nucleotide and amino acid sequences of human TNF-β are well known and are presented in Gray, (1984) Nature 312: 721-724, and accession code Swessprot P01374, each of which is incorporated herein by reference. . The amino acid sequence of human IL-10 is well known and described in Viera, et al. (1991) Proc. Natl. Acad. Sci. USA 88: 1172-1176, and accession code Swissprot P22301, each of which is incorporated herein by reference.

The complete coding sequence for human interleukin 12 mRNA is shown in Genbank accession numbers AF180563 (P40 mRNA) and AF180562 (P35 mRNA) and US Pat. No. 5,840,530, each of which is incorporated herein by reference.

Sequence information for MadCAM-1 is found in Genbank Accession No. U80016 (Leung, E., et al, Immunogenetics 46(2), 111-119(1997)), each of which is incorporated herein by reference.

Sequence information for MadCAM-1 can be found in Genbank Accession No. U43628 [Shyjan, A. M., et al, J. Immunol. 156(8), 2851-2857 (1996), each of which is incorporated herein by reference.

Sequence information for NGF is found in Genbank Accession No. M57399 (Kretschmer, P. J., et al., Growth Factors 5,99-114 (1991)), each of which is incorporated herein by reference.

Sequence information for IL-7 can be found in Genbank Accession No. J04156 [Goodwin, R. G., et al., Proc. Natl. Acad. Sci. U.S.A. 86(1), 302-306(1989), each of which is incorporated herein by reference.

Sequence information for VEGF is found in Genbank Accession No. M32977 (Leung, D. W., et al., Science 246, 1306-1309 (1989)), each of which is incorporated herein by reference.

Sequence information for TNF-R is Genbank accession number M60275 [Grey, P. W., et al. Proc. Natl. Acad. Sci. U.S.A. 87, 7380-7384 (1990), each of which is incorporated herein by reference.

Sequence information for TNF-R is Genbank accession number M63121 [see: Himmler, A., et al. DNA Cell Biol. 9, 705-715 (1990), each of which is incorporated herein by reference.

Sequence information for Fas is found in Genbank accession number M67454 (Itoh, N., et al., Cell 66 (2), 233-243 (1991)), each of which is incorporated herein by reference.

Sequence information for CD40L can be found in Genbank accession number L07414 [Gauchat, J. F. M., et al. FEBS Lett, 315, 259-266 (1992), each of which is incorporated herein by reference.

For sequence information on IL-4, Genbank Accession No. M23442 [Arai, N., et al., J. Immunol. 142 (1), 274-282 (1989), each of which is incorporated herein by reference.

Sequence information for IL-4 is Genbank Accession No. M13982 [Yokota, T., et al. Proc. Natl. Acad. Sci. U.S.A. 83 (16), 5894-5898 (1986), each of which is incorporated herein by reference.

Sequence information for the CSF is Genbank accession number M37435 [see: Wong, G. G., et al. Science 235 (4795), 1504-1508 (1987), each of which is incorporated herein by reference.

Sequence information for G-CSF is found in Genbank Accession No. X03656 (Nagata, S., et al, EMBO J. 5 (3), 575-581 (1986)), each of which is incorporated herein by reference. .

Sequence information for G-CSF is found in Genbank Accession No. X03655 (Nagata, S., et al., EMBO J. 5 (3), 575-581 (1986)), each of which is incorporated herein by reference. do.

Sequence information for GM-CSF is Genbank accession number Ml1220 [see: Lee, F., et al., Proc. Ntl. Acad. Sci. U.S.A. (13), 4360-4364 (1985), each of which is incorporated herein by reference.

Sequence information for GM-CSF is found in Genbank Accession No. M10663 (Wong, G. G., et al., Science228 (4701), 810-815 (1985)), each of which is incorporated herein by reference.

Sequence information for M-CSF is Genbank accession number M27087 [see Takahashi, M., et al., Biochem. Biophys. Res. Commun. 161 (2), 892-901 (1989), each of which is incorporated herein by reference.

Sequence information for M-CSF is found in Genbank Accession No. M37435 (Wong G. G., et al., Science 235 (4795), 1504-1508 (1987)), each of which is incorporated herein by reference.

Sequence information for LFA-3 can be found in Genbank Accession No. Y00636 [Wallner, B. P., et al., J. Exp. Med. 166 (4), 923-932 (1987), each of which is incorporated herein by reference.

Sequence information for ICAM-3 is found in Genbank Accession No. X69819, which are incorporated herein by reference.

Sequence information for ICAM-2 is found in Genbank Accession No. X15606 (Staunton, D. E., et al., Nature 339 (6219), 61-64 (1989)), which is incorporated herein by reference.

Sequence information for ICAM-1 is found in Genbank Accession No. J03132 (Staunton, D. E., et al., Cell 52 (6), 925-933 (1988)), which is incorporated herein by reference.

For sequence information on PECAM, see Genbank Accession No. M28526 [see; Newman, P. J., et al., Science 247, 1219-1222 (1990), which is incorporated herein by reference.

Sequence information for P150.95 is found in Genbank Accession No. Y00093 (Corbi, A. L., et al., EMBO J. 6 (13), 4023-4028 (1987)), which is incorporated herein by reference.

For sequence information on Mac-1, see Genbank accession number J03925 [see; Corbi, A. L., et al., J. Biol. Chem. 263 (25), 12403-12411 (1988), which is incorporated herein by reference.

For sequence information on LFA-1, see Genbank Accession No. Y00796 [see; Larson. R., et al., J. Cell Biol. 108 (2), 703-712 (1989), which is incorporated herein by reference.

Sequence information for CD34 can be found in Genbank Accession No. M81104 [Simmons, D. L. et al., J. Immunol. 148, 267-271 (1992), which is incorporated herein by reference.

Sequence information for RANTES can be found in Genbank Accession No. M21121 [Schall, T. J., et al., J. Immunol. 141,1018-1025 (1988), which is incorporated herein by reference.

Sequence information for IL-8 is Genbank accession number M28130 [see: Mukaida, N., et al., J. Immunol. 143 (4), 1366-1371 (1989), which is incorporated herein by reference.

Sequence information for MIP-lα is Genbank accession number U72395 [see Fridell, R. A., et al., J. Cell. Sci 110 (pt 11), 1325-1331 (1997), which is incorporated herein by reference.

For sequence information on E-selectone, see Genbank Accession No. M24736 [see; Bevilacqua, M. P., et al., Science 243 (4895), 1160-1165 (1989), which is incorporated herein by reference.

Sequence information for CD2 is Genbank Accession No. M14362 [Sewell, W. A., et al. Proc. Natl. Acad. Sci. U.S.A. 83,8718-8722 (1986); Proc. Natl. Acad. Sci. U.S.A.84, 7256-7256 (1987), which is incorporated herein by reference.

Sequence information for MCP-1 is Genbank Accession No. S69738 [See: Li, Y. S., et al., Mol. Cell. Biochem. 126 (1), 61-68 (1993), which is incorporated herein by reference.

For sequence information on L-selection, see Genbank Accession No. X16150 (Tedder, T. F., et al., J. Exp. Med. 170 (1), 123-133 (1989), which is incorporated herein by reference.

For sequence information on the P-selection, see Genbank accession number M25322 [see; Johnston, G. I., et al., Cell 56, 1033-1044 (1989), which is incorporated herein by reference.

For sequence information on FLT, see Genbank Accession No. X94263 [see; Mandriota, S. J., et al., J. Biol. Chem. 271 (19), 11500-11505 (1996), which is incorporated herein by reference.

Sequence information for FLT can be found in Genbank Accession No. X51602 [Shibuya, M. et al. Oncogene 5 (4), 519-524 (1990) Han, H. J., et al. Hum. Mol. Genet. 2 (12), 2204 (1993), which is incorporated herein by reference.

Sequence information for Apo-1 is Genbank Accession No. X63717 [Oehm, et al, J. Biol. Chem., (1992), 267(15), 10709-15), which is incorporated herein by reference.

Sequence information for Fas is found in Genbank Accession No. M67454 (Itoh, et al., Cell, (1991), 66 (2), 233-43), which is incorporated herein by reference.

Sequence information for TNFR-1 is found in Genbank Accession No. M67454 (Nophar, et al., EMBO J., 1990, 9 (10), 3269-78), which is incorporated herein by reference.

Sequence information for p55 is found in Genbank Accession No. M58286 (Loetscher, et al., Cell, 1990, 61, 351-359), which is incorporated herein by reference.

Sequence information for WSL-1 is found in Genbank Accession No. Y09392 (Kitson, et al., Nature, 1996, 384 (6607), 372-5), which is incorporated herein by reference.

Sequence information for DR3 is found in Genbank Accession No. U72763 (Chinnaiyan, et al., Science, 1996, 274(5829), 990-2), which is incorporated herein by reference.

For sequence information on TRAMP, see Genbank Accession No. U75381 [see; Bodmer, et al., Immunity, 1997, 6 (1), 79-88), which is incorporated herein by reference.

Sequence information for Apo-3 can be found in Genbank Accession No. U74611 [Marsters, et al., Curr. Biol., 1996, 6 (12), 1669-76), which is incorporated herein by reference.

Sequence information for AIR is found in Genbank accession number U78029, which is incorporated herein by reference.

For sequence information on LARD, Genbank accession number U94512 [see: Screaton, et al., Proc. Natl. Acad. Sci. USA, 1997, 94 (9), 4615-19), which is incorporated herein by reference.

Sequence information for NGRF is found in Genbank Accession No. M14764 (Johnson, et al., Cell, 1986, 47 (4), 545-554), which is incorporated herein by reference.

Sequence information for DR4 (TRAIL) is found in Genbank Accession No. U90875 (Pan, et al., Science, 1997, 276 (5309), 111-113), which is incorporated herein by reference.

Sequence information for DR5 is found in Genbank Accession No. AF012535 (Sheridan, et al., Science, 1997, 1227 (5327), 818-821), which is incorporated herein by reference.

For sequence information on KILLER, see Genbank Accession No. AF022386 [see; Wu, et al., Nat. Genet. 17 (2), 141-143 (1997), which is incorporated herein by reference.

Sequence information for TRAIL-R2 is found in Genbank accession number AF020501, which is incorporated herein by reference.

Sequence information for TRICK2 is found in Genbank accession number AF018657.

Sequence information for DR6 is found in Genbank accession number AF068868, which is incorporated herein by reference.

Sequence information for ICE can be found in Genbank accession numbers U13697, U13698 and U13699 [Alnemri, E. S., et al., J. Biol. Chem. 270 (9), 4312-4317 (1995), which is incorporated herein by reference.

Sequence information for VLA-1 can be found in Genbank Accession No. X17033 [Takada., et al., J. Biol. Chem. 109 (1), 397-407 (1989), which is incorporated herein by reference.

Sequence information for CD86 (B7.2) can be found in Genbank accession number U04343 (Azuma, et al., Nature. 366 (6450), 76 (1993), which is incorporated herein by reference.

Table 1a

Table 1b

Table 1c

Table 2

<110> The Trustees of the University of Pennsylvania <120> Nucleic Acid Sequences Encoding and Compositions Comprising IgE Signal Peptide and/or IL-15 and Methods for Using the same <130> UPAP0020-5000-DIv <150> US 60/478,205 <151> 2003-06-13 <150> US 60/478,210 <151> 2003-06-13 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 14 <212> PRT <213> Arificial Sequence <220> <223> chemically synthsized peptide <400> 1 Ala Met Gln Met Leu Lys Glu Thr Met Glu Glu Ala Ala Glu 1 5 10 <210> 2 <211> 9 <212> PRT <213> Arificial Sequence <220> <223> chemically synthesized peptide <400> 2 Ala Met Gln Met Leu Lys Glu Thr Ile 1 5 <210> 3 <211> 13 <212> PRT <213> Arificial Sequence <220> <223> chemically synthesized peptide <400> 3 Phe Arg Asp Val Asp Arg Phe Tyr Lys Thr Arg Ala Glu 1 5 10 <210> 4 <211> 47 <212> DNA <213> Arificial Sequence <220> <223> oligonucleotide primer <400> 4 gcccccgtcg acgccgccac catgagaatt tcgaaaccac atttgag 47 <210> 5 <211> 37 <212> DNA <213> Arificial Sequence <220> <223> oligonucleotide primer <400> 5 atcgggctcg agtcaagaag tgttgatgaa catttgg 37 <210> 6 <211> 39 <212> DNA <213> Arificial Sequence <220> <223> oligonucleotide primer <400> 6 gcccccggta ccgccgccac catggtattg ggaaccata 39 <210> 7 <211> 33 <212> DNA <213> Arificial Sequence <220> <223> oligonucleotide primer <400> 7 atcgggggat cctcaagaag tgttgatgaa cat 33 <210> 8 <211> 47 <212> DNA <213> Arificial Sequence <220> <223> oligonucleotide primer <400> 8 gcccccgaat tcgccgccac catggattgg acttggatct tattttt 47 <210> 9 <211> 49 <212> DNA <213> Arificial Sequence <220> <223> oligonucleotide primer <400> 9 agttgctgct gctactagag ttcattctaa ctgggtgaat gtaataagt 49

Claims (25)

An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence, wherein the IgE signal peptide and the non-IgE protein sequence are from an animal of the same species. The isolated nucleic acid molecule of claim 1, wherein the non-IgE protein is an enzyme or a functional fragment thereof. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence, wherein the non-IgE protein is an immunoregulatory protein or functional fragment thereof. The isolated nucleic acid molecule of claim 1, wherein the fusion protein consists of an IgE signal peptide linked to an immunoregulatory protein or functional fragment thereof. The isolated nucleic acid molecule of claim 3, wherein the IgE signal peptide and the non-IgE protein sequence are from an animal of the same species. The isolated nucleic acid molecule of claim 1 or 3, which is a plasmid. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is incorporated into a viral vector. An injectable pharmaceutical composition comprising the nucleic acid molecule of claim 1. A recombinant vaccine comprising the nucleic acid molecule according to claim 1. An attenuated live pathogen comprising the nucleic acid molecule of claim 1. A fusion protein comprising an IgE signal peptide linked to a non-IgE protein, wherein the IgE signal peptide and the non-IgE protein are from an animal of the same species. The fusion protein of claim 11, wherein the non-IgE protein is an enzyme or a functional fragment thereof. The fusion protein of claim 11, wherein the non-IgE protein is an immunoregulatory protein or functional fragment thereof. A fusion protein consisting of an IgE signal peptide linked to an immunoregulatory protein or functional fragment thereof. 15. The fusion protein of claim 13 or 14, wherein the IgE signal peptide and the non-IgE protein are from an animal of the same species. A culture comprising a cell comprising a nucleic acid molecule comprising a nucleic acid sequence comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence, the nucleic acid sequence operably linked to a regulatory factor required for expression in the cell. , In vitro cell culture wherein the IgE signal peptide and the non-IgE protein are from an animal of the same species or the non-IgE protein is an immunomodulatory protein. A cell comprising a cell comprising a nucleic acid sequence comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein consisting of an IgE signal peptide linked to a non-IgE protein sequence, the nucleic acid sequence operably linked to a regulatory factor required for expression in the cell. A method comprising culturing cells under conditions necessary for fusion protein expression for a period of time sufficient to express said fusion protein, wherein the IgE signal peptide and the non-IgE protein are from an animal of the same species, or the non-IgE protein is immune. A method of making a non-IgE protein, which is a regulatory protein. A composition comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an immunogen and a nucleic acid molecule according to claim 1. The composition of claim 18, wherein the immunogen is a pathogen antigen, a cancer-associated antigen or an antigen linked to a cell associated with an autoimmune disease. The composition of claim 18, wherein the immunogen is a pathogen antigen. The composition of claim 20, wherein the pathogen antigen is selected from the group consisting of HIV, HSV, HCV, and WNV. A composition comprising a nucleic acid molecule according to claim 1 or 3 and a nucleic acid molecule further comprising a nucleotide sequence encoding CD40L. The injectable pharmaceutical composition of claim 8 for modulating an immune response in an individual or inducing an immune response against an immunogen in an individual. The composition of claim 18 for modulating an immune response in an individual or inducing an immune response against an immunogen in an individual. The composition of claim 22, wherein the composition is for inducing an immune response against an immunogen in a subject.

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